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Review

Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives

1
Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz 7146864685, Iran
2
Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, 50134 Florence, Italy
3
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran
4
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115111, Iran
5
Cancer Science Institute of Singapore, National University of Singapore, Singapore 637551, Singapore
6
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 637551, Singapore
7
Department of Biology, Faculty of Sciences, Shahid Bahonar University, Kerman 7616914111, Iran
8
Student Research Committee, Kerman University of Medical Sciences, Kerman 7619813159, Iran
9
Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman 7616911319, Iran
10
Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Istinye University, Sariyer, Istanbul 34396, Turkey
11
Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman 7619813159, Iran
12
NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore 637551, Singapore
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2021, 22(21), 11321; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111321
Submission received: 6 October 2021 / Revised: 17 October 2021 / Accepted: 17 October 2021 / Published: 20 October 2021
(This article belongs to the Special Issue Feature Annual Reviews in Molecular Genetics and Genomics 2021)

Abstract

:
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins (Cas) is an adaptive immune system in archaea and most bacteria. By repurposing these systems for use in eukaryote cells, a substantial revolution has arisen in the genome engineering field. In recent years, CRISPR-Cas technology was rapidly developed and different types of DNA or RNA sequence editors, gene activator or repressor, and epigenome modulators established. The versatility and feasibility of CRISPR-Cas technology has introduced this system as the most suitable tool for discovering and studying the mechanism of specific genes and also for generating appropriate cell and animal models. SOX genes play crucial roles in development processes and stemness. To elucidate the exact roles of SOX factors and their partners in tissue hemostasis and cell regeneration, generating appropriate in vitro and in vivo models is crucial. In line with these premises, CRISPR-Cas technology is a promising tool for studying different family members of SOX transcription factors. In this review, we aim to highlight the importance of CRISPR-Cas and summarize the applications of this novel, promising technology in studying and decoding the function of different members of the SOX gene family.

1. Introduction

1.1. CRISPR-Cas System

The CRISPR-Cas is an adaptive immune system used by bacteria and archaea to protect themselves from foreign nucleic acids invasion, such as phages or plasmids [1,2,3]. In 2012, for the first time, the CRISPR-Cas system was demonstrated to be a promising genome editing tool to be used for selective gene manipulation in both in vitro and in vivo models (Figure 1) [4,5]. Compared to genome editing tools based on protein-DNA interaction such as meganucleases, ZFNs, and TALENs, the technology of CRISPR-Cas is more versatile and feasible because it relies on base pairing of nucleic acids; and the required guide RNA (gRNA), the complex of crRNA and tracrRNA could be easily engineered as a single transcript, avoiding the need for custom synthesis, purification, and validation of targeted DNA-binding proteins [5,6,7].
Native CRISPR-Cas systems are classified into two main classes and subcategorized into six types of Cas effector proteins. CRISPR-Cas9, classified into class-II and type II, is the most common system widely used in several CRISPR-based genome editing approaches [8]. Moreover, two other popular single effector Cas proteins in class-II are (i) Cas12a, which is subcategorized in type V and, unlike the natural Cas9, recognizes T-rich PAM, by a single gRNA [9,10] and (ii) Cas13, which is classified in type VI and is able to precisely target the RNA [11]. To date, several strategies are available to deliver the CRISPR-Cas machinery in vitro and in vivo.
The delivery systems are needed for biomedical applications of CRISPR-Cas [6,12]. Viral vectors, comprising adenoviruses, adeno-associated viruses (AAVs), and retroviruses, are very efficient delivery systems [12,13]. Moreover, viruses are extensively used for cancer therapy due to their ability to preferentially infect cancer cells in an active proliferative status, exploited in the commonly called oncolytic therapy [14]. Other less widely used methods are combining the exogenous form of the Cas9 with the selected gRNA, a formulation called ribonucleoproteins (RNPs), transferable into cells by lipid-mediated delivery [15], exosomes-derived vesicles [16], nano-formulations such as gold nanoparticles [17], copper sulfide nanoplatform [18], dendrimers [19], apoferritin [20], supramolecular polymers [21] or nanoclews [22]. The advantages of these nonviral delivery systems include low immunogenicity and expense, simple scalability, and safety [23,24,25,26,27,28,29,30,31,32,33]. All the above-described systems have expanded the possibilities to deeply study the molecular mechanisms and help decode unknown functions of several important cell proteins [34]. The SOX gene family are associated with several biological functions, from stemness to carcinogenesis, and by using CRISPR-Cas genome editing tools, the exact mechanism and function would be more reliably appreciated.

1.2. SOX Proteins

The SOX (SRY homology box) proteins affect stem cell function and fate by regulating the expression of genes involved in self-renewal and multipotency [35,36,37]. These proteins are overexpressed in many different tumors [38]. SOX proteins are able to bind to the DNA sequence motif ATTGTT, trigger conformational changes, and bend DNA specifically [39]. These proteins as ‘pioneer’ factors recruiting non-pioneer transcription factor (TFs) drive cell fate conversions [40]. This protein family includes 20 members, which mainly share a conserved DNA-binding element HMG domain, a transcriptional master regulator of virility [41]. SOX proteins as lineage-associated TFs are classified into different categories (from SOXA to H) according to homology within the HMG domain (Figure 2) [42].
Post-translation modifications of SOX proteins including phosphorylation, methylation, ubiquitylation, acetylation, and SUMOylation have been reported and represent an annotated function. Therefore, targeting the enzymes that catalyze these modifications may affect therapeutic strategies for human diseases [43]. Some published review articles have discussed the diverse functions of SOX proteins across cancer, stem cells, and development [35,44,45,46]. For example, SOX8 was a master regulator for sense organ cell reprogramming [47] and SOX15 determined as a oocyte-enriched reprogramming factor [48].
In this review, we aim to summarize recent advancements in studying different members of the SOX gene family by using CRISPR-Cas genome editing tools. The versatility and feasibility of CRISPR technology introduced this system as a promising tool for uncovering unknown mechanisms and drawing reliable signaling pathways. SOX genes are extremely important factors, especially during development (Figure 3) [49] and cancer initiation, progression, invasiveness, and metastasis (Figure 4) [46,50]. Compared to other genome editing tools, CRISPR-Cas technology is easily programmable, cost-effective, and could be efficiently applied to study different SOX genes and treat SOXopathies [49].

2. SOX2 Involvement in Cancer and Stem Cell Fates

SOX2 is currently the most studied member of the large SOX family, often interacting with a series of co-factors such as OCT3/4 and PAX6 [51,52] through its C-terminus domain, while its N-terminus, having the HMG-domain, the nuclear localization sequence and the nuclear export sequence, plays a fundamental role in the subcellular distribution [53,54]. The HMG domain in particular has been characterized in deep and a DNA recognition consensus has been defined for SOX2 (i.e., CCCATTGTTC in man and CTTTGTC in mouse) [40,55]. Although the TTGT element is the preferred recognition motif for all SOX proteins, SOX2 is able to keep in contact with several transcription factors in an unspecific and promiscuous manner [56]. It is interesting to note that SOX2 is able to up-regulate itself and exploit an autoregulatory feed-forward mechanism [57]. Interestingly, the entire SOX2 gene further falls into the intron of a much greater product, which is called SOX2OT (SOX2 overlapping transcript) [58]. Indeed, when planning a SOX2 knock-out (KO) experiment, it should be taken under consideration that every SOX2 manipulation obviously involves potential side-effects on SOX2OT [59]. SOX2OT plays an important role in carcinogenesis by promoting tumor cell proliferation, invasion, migration, and growth and suppressing apoptosis mainly through the regulation of some cancer stem cell (CSC) factors such as OCT4, NANOG, ALDH1, CD44, and CD133 [60,61]. Moreover, it was established that SOX2OT is also capable to bind miR-200 family members to regulate SOX2 expression. SOX2 is not the only member of the SOX family regulated by SOX2OT as it was reported that it is able to modulate the mRNA and protein expression of SOX3 as well [62]. SOX2OT was demonstrated to promote epithelial-mesenchymal transition and stemness in several kinds of cancer cells [63,64,65]. SOX2OT is also involved in the inhibition of the JAK/STAT signaling pathway [62]. While two gene-proximal enhancers, SOX2 regulatory region 1 (SRR1) and SRR2 were previously described to act as cis-regulator for SOX2 expression [66], Zhou et al. identified three novel enhancers, i.e., SRR18, SRR107, and SRR111, which form a chromatin complex with the SOX2 promoter in embryonic stem cells (ESCs) [67]. Moreover, a 13 kb-long super-enhancer was described to be located 100 kb downstream of SOX2 in mouse ESCs, which may interact with OCT4, SOX2, and Nanog as trans-acting factors to enhance SOX2 expression via DNA looping. Li et al. used a double-CRISPR genome editing approach to delete the entire super-enhancer sequence and demonstrate that it is responsible for over 90% of SOX2 expression [68]. Deleting the core of the CCCTC-binding factor (CTCF) binding site by CRISPR-Cas9 editing tool in the SOX2 super-enhancer resulted in cohesin recruitment loss and disrupting the formation of chromatin loops, which in turn reduced SOX2 expression [69].
SOX2 is widely known as a master orchestrator in all reprogramming applications. Moreover, SOX2 collaborates with several co-factors such as OCT4, KLF4, and cMYC [70] and enables the derivation of human or murine-induced pluripotent stem cells (iPSCs) from terminally differentiated somatic cells. Such phenomenon was partially observed in vivo, where SOX2 plays key roles in the stem potential of the inner cell mass of the blastocyst [71] and neural cell lineages formation [72]. Moreover, SOX2 is orchestrating the development of the gastrointestinal tract, where its expression is associated with the engulfing foregut and derived endodermal structures from which the esophagus and anterior stomach evolve [73]. SOX2 balancing is important for tissue homeostasis and therefore its aberrant expression is often associated with various forms of cancers [74]. In fact, SOX2 induces the acquisition of stem-like features and in some cases, generates CSCs (Figure 5) [75].
In particular, SOX2 induction has been reported at early stages in breast or ovarian cancers and is associated with disease progression, metastasis, and relapse [77,78]. A high expression level of SOX2 is associated with increased cell motility and metastasis in glioblastoma [79]. Whereas in gastric cancer, Helicobacter pylori infections have influenced SOX2 expression, activated PTEN, and consequently inhibited PI3K/AKT-driven cell cycle progression and apoptosis [80]. Moreover, it is elucidated that microenvironmental factors might play an important role in SOX2 regulation in cancer [81]. Indeed, extracellular acidosis was demonstrated to increase SOX2 expression in melanoma, confirming that SOX2 is also able to influence cancer cell metabolism profile to a more oxidative phenotype through the hypoxia-inducible factor 1-α (HIF1α) pathway [74]. SOX2, which is induced by an acidic microenvironment, was demonstrated to enhance several OxPhos-related genes and thus, its depletion led to a more glycolytic profile, negatively regulating PGC1α and inducing the switch of MCT genes from type 1 to type 4. Indeed, chemoresistant cancer cells, often characterized by high levels of SOX2, are commonly more prone to exploit the oxidative metabolism [82]. In normoxia, HIF1α and SOX2 are inversely correlated, reprogramming cancer cells towards an OxPhos profile, while under hypoxic conditions, as well as in acidosis-exposed cells, the increased lactate production may promote HIF1α stabilization reducing PGC1α towards a glycolytic re-conversion. Indeed, such a metabolic switch is often associated with enhanced drug resistance and metastatic ability [83,84]. By emerging CRISPR-Cas9 genome editing technology, several research lines have been established to better understand the importance of SOX2 in cell differentiation, the acquisition of stem characteristics, and tumor progression (Table 1) [85,86,87].

SOX2 Gene Editing Mediated by CRISPR

SOX2 plays a major role in both tumorigenesis and embryogenesis, especially during the development and differentiation of the neuroectodermal layer. Yang et al. demonstrated that CRISPR-Cas9-mediated KO of ATF1 significantly up-regulates neuroectoderm genes, SOX2 and PAX6, in human embryonic stem cells (hESCs). However, the overexpression of ATF1 suppressed neuroectodermal differentiation. In line with these premises, they indicated that SOX2 induction is pivotal for the up-regulation of PAX6 and SOX1, and introduced ATF1 as a negative regulator for SOX2 expression [88]. Cheng et al. exploited the PC transposon system to KO SOX2 in neural progenitor cells by in utero electroporation (IUE) with promising results. Indeed, SOX2 depleted as early as three days post-IUE, whilst expressions of SOX1 and PAX6 remained intact, demonstrating no off-target effects [89]. Moreover, they also proved that both the wild-type Cas9 and the Cas9n exert the gene-editing with comparable KO efficiency. It is also elucidated that knocking out SOX2 impaired the induction of the neural progenitor gene, Hes5, in mouse and chick embryos and the subsequent commitment to the neuronal lineage. In fact, SOX2 promotes the neurogenic domain formation in the nasal epithelium, establishes, maintains, and expands the neuronal progenitor pool by decreasing Bmp4 and up-regulating Hes5 expression. Therefore, SOX2 acts as a negative regulator for Bmp4 expression [90]. As both the neural crest and derma originated from the ectodermal layer, in some cases, brain tumors and melanoma share several features. However, melanoma cells mainly have a high expression level of SOX2 while it is not observed in neural crest stem cells [91]. To date, the role of SOX2 has been controversial in melanoma. Although it is reported that SOX2 may start the tumor initiation process via CDK1 in melanoma [92], knocking-out of SOX2 by CRISPR-Cas9 does not affect melanoma progression and metastasis [91]. Maurizi et al. demonstrated that SOX2 is required for osteosarcoma initiation and development in a mouse tumor model and is essential for survival and proliferation. They indicated that SOX2 inhibition by CRISPR-Cas9 in osteosarcoma cells decreases viability and proliferation of both CSC and non-CSC populations. Furthermore, it is indicated that the overexpression of YAP rescues cells from the lethality caused by SOX2 inactivation [93]. The loss of SOX2 is sufficient to maintain a seminoma-cell fate of seminomatous TCam-2 cells and after in vivo injection for about six weeks, these cells have been reprogrammed to an embryonal-like status. Moreover, knocking-out of FOXA2 strengthened such effect up to 12 weeks [94]. CRISPR/Cas9 was also exploited to generate a luciferase knock-in (KI) system under the control of the SOX2 promoter in HEK293T cells, demonstrating to be a novel and useful tool to study the transcriptional regulation of SOX2 [89]. Similarly, Balboa et al. provided a fluorescent marker of SOX2 endogenous expression through the knocking-in of a T2A fused nuclear tdTomato reporter before the stop codon of the SOX2 gene coding sequence by CRISPR-SaCas9 [95]. To date, many fluorescent systems are available to evaluate SOX2 endogenous expression rapidly and in a real-time manner in several cell lines to better understand how it is spatially and temporally regulated during embryogenesis and neural differentiation [96]. Yang et al. generated mice with a tag or a fluorescent reporter construct in Nanog, SOX2, and OCT4 genes through a one-step procedure by co-injection of gRNAs and Cas9 mRNA directly into zygotes, demonstrating that with such methodology the risk of off-target mutation is significantly reduced [97]. Mei et al. exploited CRISPR-dead Cas9 (dCas9) system to induce Yamanaka’s factors (OCT4, SOX2, KLF4, and MYC) in a luminal breast cancer cell line with an innovative multiplexing system. Indeed, they designed particular tRNA-gRNA architecture in order to allow the endogenous cell tRNA-processing system to precisely cleave both ends of the tRNA precursor and thus release the gRNAs. Such an approach led them to gain a stable cell strain characterized by increased invasion, proliferation and stemness features, with a similar drug response pattern of HER2 positive cells [98]. Moreover, Chang et al. used CRISPR-dCas9 to induce SOX2 expression in the rat cornea. They reported that the activation of SOX2 reduces the opacity and the thickness of the central cornea by increasing cell viability and proliferation of corneal endothelial cells, which normally are not able to regenerate after a wound or a disease (Figure 6) [99].

3. SOX2/3 Contribution in Regenerative Medicine

Of particular interest is the CRISPR application on the little salamander, called Axolotl (Ambystoma mexicanum), which is to date the only tetrapod that functionally regenerates all cell types of the limb and spinal cord. For this reason, it represents an important animal model for regenerative medicine studies [103]. Fei et al. exploited both TALEN and CRISPR technologies to KO SOX2 and demonstrated that its expression is the key for spinal cord regeneration while it did not affect larval viability and development. On the other hand, they also indicated that SOX3 expression is fundamental during development, but not for regeneration after tail amputation [100]. By using the HDR repair pathway via CRISPR-Cas9, they also successfully inserted a fluorescent reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 cassette into SOX2 and Pax7 genomic loci in the axolotl animal model. In these regards, it was elucidated that PAX7-positive satellite cells are the major contributing source in the myogenesis process during axolotl limb regeneration, while SOX2-positive cells are mainly located in the central nervous system, the lens, the head/tail lateral line neuromasts, and the spinal cord [104]. Knocking-out of SOX2 has also been performed by in vivo injection of Cas9 protein–gRNA complexes into the spinal cord lumen of the axolotl, with subsequent electroporation [105]. Such an approach was incredibly efficient through protecting Cas9 from typical RNase activity in the cerebral spinal fluid, which normally prevents the electroporation of unprotected mRNAs [106]. The major challenge using human pluripotent stem cells in regenerative therapy is the risk of teratoma formation due to contamination of undifferentiated stem cells. To overcome this limitation, using a suicide gene for killing undifferentiated stem cells seems a promising strategy to provide a safety control before transplantation of stem cell-derived products. Hence, Wu et al. used the CRISPR-Cas9 editing tool to KI the iC9 suicide gene into the endogenous SOX2 locus in the hESC-H1. With this strategy, they demonstrated that undifferentiated H1-iC9 cells were committed to apoptosis by iC9 inducer AP1903, whilst differentiated cell lineages including hematopoietic cells, neurons, and islet beta-like cells were not affected [101].

4. SOX9 Gene Editing Mediated by CRISPR

SOX9 has shown a great impact on the specification, differentiation, and maintenance of various cell types during cell development [107,108]. Various developmental processes such as sex determination, pancreas development, and chondrogenesis are associated with the expression of SOX9. Several studies reported the pathological consequences of SOX9 mutations. For example, it has been shown that the SOX9 mutation leads to campomelic dysplasia. This disorder is characterized by bowed and shortened long bones, the bell-shaped thorax, and respiratory distress. Abnormal SOX9 is often accompanied by sexual disorders [109,110,111]. In most mammals, SRY, a key factor for sex determination, up-regulates SOX9, which is important for testis formation [112,113]. It has been shown that testis-specific enhancer of SOX9 core (TESCO) acts as SOX9 enhancer and provides the binding site for SRY. In addition to TESCO, several other enhancers are associated with sex determination in mammals [114,115]. Totally, transcriptional activator SOX9 has been studied in several investigations and its importance has been demonstrated by different approaches such as CRISPR-Cas9 tools. It is revealed that far upstream of SOX9, there is a specific sequence, which is named the XY sex reversal region (XYSR). In order to identify a responsible sequence in XYSR, Ogawa et al. employed the CRISPR-Cas9 system to generate mutant mice with different deletions in XYSR. When the whole or partial sequence of XYSR was deleted, male to female sex reversal occurred. Interestingly, this sequence includes a gonad enhancer for SOX9. This study showed the application of CRISPR-Cas9 system for identification of critical sequences in sex determination via SOX9 [116]. In addition to TESCO, the role of a 3.2 kb testis specific enhancer of SOX9 (TES) in sex determination has been shown in several studies. Using CRISPR-Cas9 gene editing system for deleting TES or TESCO in XY fetal gonads, a reduction in SOX9 expression level was observed. The results of this study indicated that TES and TESCO are substantial elements for regulation of SOX9 transcription levels. However, the results showed that these elements are not the sole factors involved in sex determination via SOX9 [115].
The application of the CRISPR-Cas9 gene-editing platform is not limited to understanding the mechanism of sex determination through SOX9. There are several reports indicating that SOX9 is a gastrointestinal stem cell marker with oncogenic properties in tumor development. The up-regulation of SOX9 has been reported in various premalignant tumors [117,118]. On the other hand, the Hippo signaling pathway and YAP1, as its co-activator, were reported to play a significant role in the development of gastric cancer. It has been demonstrated that the transcription of SOX9 is regulated by the interaction between YAP1 and TEAD proteins at the SOX9 promoter, which leads to the induction of CSC properties [119]. Also, there are several reports on the role of peroxisome proliferator-activated receptors (PPARs) in gastric cancer progression [120]. Knocking down of PPARδ in cell line models decreased the formation of tumorspheres as well as invasion via the reduction of SOX9 expression. Disrupting YAP1 or SOX9 by CRISPR-Cas9 gene-editing platform reduced PPARδ-mediated oncogenic functions. In other words, the poor clinical outcome of gastric cancer patients is correlated with high levels of YAP1 or SOX9. Furthermore, the transcription of SOX9 is promoted by the formation of the PPARδ/YAP1 complex, which was shown by the CRISPR-Cas9 gene-editing system. [121].
SOX2-dependent activation of Wnt signaling in tamoxifen-resistant breast cancer cells leads to the increase of CSC content. Moreover, it has been shown that high levels of SOX9 are associated with shorter survival and poor clinical outcome in breast cancer patients [122,123]. In a study conducted by Domenici et al., the growth of tamoxifen-resistant breast tumors in an in vivo model reduced after knocking out of SOX9 by using CRISPR-Cas technology. According to the results of this study, SOX2–SOX9 signaling axis can act as a key factor in controlling the luminal progenitor cell content and is essential for the activity of Wnt signaling pathway. Knocking out of SOX9 by the CRISPR-Cas system has represented it as a potential therapeutic target in breast cancer [124].
Another application of CRISPR-Cas systems is the generation of various differentiated cells from human pluripotent stem cells. The differentiated cells could be generated from various cells, including ESCs and iPSCs [125]. It is indicated that astrocytes can rapidly (in 4 to 7 weeks, while conventional methods take 3 to 6 months) be generated from hESCs when the expression level of transcription factors including NFIA or NFIA plus SOX9 had been elevated by using CRISPR-Cas9 technology. This simple and fast method has provided the great opportunity to investigate the biological properties of astrocytes as well as their role in several diseases processes [125,126].

5. CRISPR-Cas Editing of Other SOX Genes

It has been shown that the pluripotency and differentiation of ESCs are highly dependent on RYBP (Ring1 and YY1 Binding Protein). Differentiation of ESCs to myocardial and neural cells could be disrupted by depletion of RYBP. To investigate the role of RYBP in neural differentiation, CRISPR-Cas9 genome editing technology was employed to generate an RYBP homozygous KO murine ESCs containing SOX1-GFP reporter. The generated cell line could be used for the investigation of the ESCs differentiation into neurons as well as the study of molecular mechanisms of neurogenesis and drug screening [127].
The role of SOX2 and SOX3 genes in the development of mouse testes and brain has been investigated to show their functional equivalency [128]. By using the CRISPR-Cas9 system, these two genes were mutated to demonstrate their functions are identical or different. The replacement of SOX3 with SOX2 revealed that the increased expression of SOX2 functionally rescues the defects related to the depletion of SOX3 in the development of pituitary and testes and restores phenotypes associated with SOX3-null mice. These results demonstrated the equivalent functions of SOX2 and SOX3 for brain and testis development [128]. Likewise, for studying the role of SOX2 and SOX3 in otic/epibranchial placode induction, Gou et al. used CRISPR-Cas9 technology to generate mutant alleles of SOX2 and SOX3. Their results demonstrated redundant functions of these genes in the production of otic and epibranchial tissue [129]. Moreover, this investigation also elucidated that the loss of SOX3 could be replaced by SOX2 expression to rescue placodal deficiencies. The use of the CRISPR-Cas9 system in a zebrafish model revealed the cooperation of SOX2 and SOX3 in the regulation of otic/epibranchial placode induction [129].
However, the role of SOX3 is not limited to the brain, testis, or otic/epibranchial placode induction. Hong et al. investigated the molecular mechanism of folliculogenesis for the generation of female gametes. They generated SOX3 KO zebrafish lines by using the CRISPR-Cas9 system to investigate the pathways involved in ovarian steroidogenesis and apoptosis. The involvement of SOX3 in these cellular processes was shown in SOX−/− ovaries by up-regulation of apoptotic pathways in such cells while ovarian steroidogenesis was down-regulated [130]. SOX3 KO also resulted in the retardation of follicle development. Moreover, it is indicated that SOX3 could bind to the promoter of cyp19a1a and enhance 17β-estradiol synthesis, which in turn prohibits apoptosis in follicle development [130].
Another interesting member of the SOX family is SOX4, which has shown a great impact on cellular development and differentiation. SOX4 has demonstrated significant transcriptional activation roles as well as suppression functions alone or together with other transcription factors. Several reports indicated the importance of SOX4 expression in bladder cancer development. However, the exact role of SOX4 in bladder cancer tumorigenesis had not been elucidated. To find SOX4-regulated genes in the progression of bladder cancer, the CRISPR interference (CRISPRi) method was employed to suppress the expression of SOX4 in specific bladder cancer cell lines. When the expression of SOX4 was restored by using lentiviral vectors, the targeted cells were rescued, and this experiment showed the pivotal role of SOX4 in tumorigenesis. On one hand, restoration of SOX4 expression increased invasiveness of cancer cells whereas this characteristic was decreased in SOX4 KO cells. On the other hand, proliferation and migration properties did not change significantly in these cells, showing that SOX4 has no notable impact on these processes. Furthermore, gene expression profiling elucidated that there is a negative correlation between SOX4 and WNT5a expression levels, suggesting that low expression levels of SOX4 result in higher levels of WNT5a, which is associated with decreased invasion phenotype. Therefore, it could be concluded that the invasion of bladder cancer cells is regulated by SOX4 through the repression of WNT5a [131].
SOX6 is another member of the SOX family, which has shown a substantial role in β-thalassemia. Red blood cell destruction could be decreased by the reactivation of fetal γ-globin. SOX6, as a negative regulator, binds to the γ-globin promoter, and silences the expression of fetal hemoglobin. Therefore, reactivation of γ-globin could be considered as a therapeutic approach to ameliorate the symptoms of β-thalassemia [132,133,134]. Silencing SOX6 expression by using CRISPR-Cas9 technology revealed that γ-globin mRNA level increased. This finding indicated that inhibiting SOX6 expression by using CRISPR-Cas9 technology could be considered as a therapeutic strategy for the treatment of β-thalassemia patients [135].
Great attention has been directed to SOX10 due to its important roles in the inner ear and embryonic development, particularly in neural crest cells and neural crest derivatives such as melanocytes [136]. Several reports indicated that SOX10 mutations have various characteristic phenotypes, including severe hearing loss and pigmentary disturbance [137,138]. In literature, it is revealed that mutation in SOX10 orthologous has an association with Waardenburg diseases. CRISPR-Cas technologies have shown promising improvements in generating animal models, which are extremely valuable in studying human unknown mutations. It is indicated that CRISPR-Cas9 technology led to successful results to generate gene-modified pigs harboring precise genetic mutation [139]. The chick embryo provides a great opportunity for such experiments due to the low cost and ease of manipulation. In this line, to KO SOX10 as a key transcription factor in neural crest development, CRISPR-Cas9 technology was employed to silence this gene in the early chick embryo. The successful loss-of-function in chick embryos could be used in several developmental processes, including dissection of gene regulatory interactions [140]. Neural crest stem cells have a great potential for differentiation into various cell types. Since SOX10 is produced in early neural crest progenitors, the CRISPR-Cas9 genome editing tool was used to generate SOX10-Nano-lantern (NL) reporter hiPSCs. In this investigation, neural crest cells were purified from hips with an NL KI reporter. Unlike conventional SOX10-reporter lines, these cells achieved bicistronic expression of NL and SOX10 gene. The SOX10-expressing cells showed self-renewal properties as well as great potential for differentiation into neural crest derivatives [141]. In another study, a non-disruptive SOX10 KI reporter was generated using the CRISPR-Cas9 genome editing tool in rat ESCs to generate both in vitro and in vivo reporter models. Recently, it was indicated that SOX10 has the potential for visualization and isolation of precursor and mature oligodendrocytes from postnatal animals. On the other hand, rats have shown several advantages rather than transgenic mouse lines due to the relative ease of surgical procedures and superiority of demyelinating lesions in rat models [142,143]. In addition, rat models are more suitable for cognition assays. The successful germline transmission provided a platform to generate animal models [144].
SOX10 has been considered as an oligodendrocyte lineage master regulator gene that could be used for reprogramming fibroblast cells to oligodendrocyte progenitor-like cells. In an investigation conducted by Matjusaitis et al., SOX10 along with Olig2, and Nkx6-2 were delivered to the target cells to enhance the differentiation of neural stem cells. Delivery of these three key oligodendrocyte lineage master regulatory genes resulted in reprogramming mouse embryonic fibroblasts to oligodendrocyte progenitor-like cells [145].
The association of the SOX family with the ability of the peripheral nervous system (PNS) for regeneration following injury has been shown via activation of the transcription factor SOX11 [146]. Perry et al. also confirmed that a regulatory network orchestrates the regeneration program in PNS following injury [147]. The regeneration-associated genes (RAGs) are members of the transcriptional response to injury and result in the synthesis of adhesion molecules and neuropeptides as well as cytoskeletal elements and cytokines [148]. The most important RAGs include transcription factors such as Jun, Atf3, and SOX11. Moreover, it is found that long noncoding RNAs (lncRNAs) play a key role in the regeneration of neurons. It is demonstrated that one of the lncRNAs, Silc1, led to neuroregeneration via the activation of SOX11 [147].
The other member of the SOX family is SOX17, which has shown a great impact on endoderm development. In developmental biology studies, mouse models are commonly used to study the function and molecular mechanism of specific genes in developmental processes. In a study carried out by Suzuki et al., CRISPR-Cas9 technology was used to generate SOX17-2AEGFP (endoderm marker), Otx2-2A-tdTomato (ectoderm marker), and T-2A-TagBFP (mesoderm marker) bicistronic reporter KI mouse models. These mouse models enable researchers to visualize the endodermal, ectodermal, and mesodermal tissues during gastrulation [149].
Two genome-wide association studies and a meta-analysis analysis were carried out to identify the genetic determinants of risk in pulmonary arterial hypertension (PAH). The results revealed that the risk variants near SOX17 change gene regulation through a lineage-specific enhancer, which is active in endothelial cells. When this enhancer was inactivated by using CRISPR-Cas9 technology, a significant reduction in SOX17 expression was observed. These results confirmed the association of genetic variation in the enhancer near SOX17 with PAH. In this line, more attention is needed to find the impairment of SOX17 function that results from the genetic variation at loci the enhancer near SOX17.
Reduced fertility on female mice has been reported following the haploinsufficiency of SOX17. Also, infertility has been observed in mice with ablation of SOX17 in progesterone receptor promoter (Pgr)-positive cells due to the lack of uterine glandular structures [150]. Based on several investigations, SOX17 acts as a downstream target of the Pgr-Gata2-dependent transcription network. Wang et al. reported that ablation of SOX17 could impair leukemia inhibitory factor and Indian hedgehog homolog (IHH) signaling pathway and result in embryo implantation failure. Deleting SOX17-binding region, 19 kb upstream of the Ihh locus, by using CRISPR-Cas technology reduced Ihh expression which in turn resulted in the pregnancy impairment. These results showed that SOX17 regulates endometrial epithelial-stromal interactions and acts as a key regulatory element necessary for endometrial epithelial gene expression (Table 2) [151].

6. Conclusions

Genome editing tools during recent years have had a great impact on scientific research and therapeutic approaches. The CRISPR-Cas systems, winner of Nobel Prize in 2020, are the most groundbreaking technology in the field of genome editing and have revolutionized the insights towards establishing novel therapies for improving human health. By utilizing CRISPR-Cas systems, we can easily introduce various kinds of modifications such as targeted editing of DNA or RNA sequence, up-regulating or down-regulating specific genes, and even reprogramming epigenetic status in target cells. CRISPR-Cas-based tools have emerged as a powerful gene modulator mostly in in vitro studies. In this line, it would be extremely crucial to evaluate the efficiency of CRISPR-based tools in different in vivo applications. Currently, there are several crucial challenges, including off-target effects and promising methods for delivering the CRISPR-Cas system into target cells that need to be addressed precisely. Delivering the RNP form of the CRISPR-Cas system has shown promising results rather than plasmid DNA or mRNA. Recently, synthetic nanoparticles have been used broadly for delivering CRISPR-Cas systems in both in vitro and in vivo studies. However, it seems urgent that long-term studies should be performed to validate the safety of the components which are utilized in these cases. Recently, it was reported that the CRISPR-Cas clinical trial was successful for sickle cell disease and β-thalassemia. This finding could bring promising hope for ex vivo gene editing strategies and their use in clinical trial approaches [156]. CRISPR-Cas systems are powerful tools for discovering and studying genes such as the SOX family of transcription factors. SOX factors play important roles in development and stem cell biology. Generating appropriate cell and animal models gives the opportunity to address fundamental questions about the roles of SOX factors in the development process compared with their impact on tissue homeostasis and regeneration. The versatility and feasibility of CRISPR-Cas systems help the researchers to use these tools in studying the mechanisms of SOX factors in biological processes. The combination of CRISPR-based screening systems with available expression and ChIP-seq data could discover unknown partners for SOX factors that would help to generate more appropriate models and cell lineages from cultured pluripotent or differentiated cells. SOX factors such as SOX17 and SOX9 seem also play supporting roles in initiating human cancers by providing primitive stem-cell-like states. CRISPR-Cas systems could be applied for decoding the real function of SOX factors in human cancers as well.
In summary, CRISPR-Cas tools have revolutionized research and therapeutic approaches in the field of genome engineering. During recent years, CRISPR-based tools have shown promising potential for use in discovering and studying the mechanisms of human genes. SOX genes are pivotal factors during development processes in stem cell biology. By using CRISPR-Cas technologies, the exact function of SOX factors in these processes could be elucidated and might lead to establish novel therapeutic strategies for human diseases and cancers.

Author Contributions

R.M. and A.P.K. conceptualized this idea; Draft preparation and figures: A.D., A.B., H.B., E.H.C.L.; M.H., H.S.F. and A.Z.; Writing and editing: H.B., E.H.C.L., A.Z., R.M., and A.P.K.; All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by a grant from the Singapore Ministry of Education Tier 2 (MOE-T2EP30120-0016) to A.P.K. A.P.K. is also supported by the National Medical Research Council of Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiative to Cancer Science Institute of Singapore, National University of Singapore. A.B. was supported by a postdoctoral fellowship of the Italian Foundation for Cancer Research (FIRC).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

CRISPRClustered Regularly Interspaced Short Palindromic Repeats
CasCRISPR-associated proteins
crRNACRISPR RNA
tracrRNATrans-activating crRNA
DSBDouble-stranded breaks
sgRNA/gRNASingle guide-RNA
ZFNZinc-finger nuclease
TALENTranscription activator-like effector nucleases
PAMProtospacer Adjacent Motif
SOXSRY homology box
KOKnock-out
KIKnock-in
CSCCancer Stem Cell
hESCHuman Embryonic Stem Cell
iC9iCaspase 9
dCas9Dead Cas9
TESCOTestis-specific enhancer of SOX9 core
XYSRXY sex reversal region
PPARPeroxisome proliferator-activated receptor
RYBPRing1 and YY1 Binding Protein
CRISPRiCRISPR inhibitor
PNSPeripheral nervous system
RAGRegeneration-associated genes
lncRNALong noncoding RNA
OxPhosOxidative phosphorylation

References

  1. Bayat, H.; Omidi, M.; Rajabibazl, M.; Sabri, S.; Rahimpour, A. The CRISPR growth spurt: From bench to clinic on versatile small RNAs. J. Microbiol. Biotechnol. 2017, 27, 207–218. [Google Scholar] [CrossRef]
  2. Bayat, H.; Naderi, F.; Khan, A.H.; Memarnejadian, A.; Rahimpour, A. The impact of crispr-cas system on antiviral therapy. Adv. Pharm. Bull. 2018, 8, 591. [Google Scholar] [CrossRef]
  3. Wiedenheft, B.; Sternberg, S.H.; Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012, 482, 331–338. [Google Scholar] [CrossRef]
  4. Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 1987, 169, 5429–5433. [Google Scholar] [CrossRef] [Green Version]
  5. Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821. [Google Scholar] [CrossRef]
  6. Mohammadinejad, R.; Sassan, H.; Pardakhty, A.; Hashemabadi, M.; Ashrafizadeh, M.; Dehshahri, A.; Mandegary, A. ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line. Bratisl. Lek. Listy 2020, 121, 31–36. [Google Scholar] [CrossRef]
  7. Mohammadinejad, R.; Biagioni, A.; Arunkumar, G.; Shapiro, R.; Chang, K.-C.; Sedeeq, M.; Taiyab, A.; Hashemabadi, M.; Pardakhty, A.; Mandegary, A. EMT signaling: Potential contribution of CRISPR/Cas gene editing. Cell. Mol. Life Sci. 2020, 77, 2701–2722. [Google Scholar] [CrossRef]
  8. Koonin, E.V.; Makarova, K.S.; Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 2017, 37, 67–78. [Google Scholar] [CrossRef]
  9. Bayat, H.; Modarressi, M.H.; Rahimpour, A. The conspicuity of CRISPR-Cpf1 system as a significant breakthrough in genome editing. Curr. Microbiol. 2018, 75, 107–115. [Google Scholar] [CrossRef]
  10. Zetsche, B.; Gootenberg, J.S.; Abudayyeh, O.O.; Slaymaker, I.M.; Makarova, K.S.; Essletzbichler, P.; Volz, S.E.; Joung, J.; Van Der Oost, J.; Regev, A. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015, 163, 759–771. [Google Scholar] [CrossRef] [Green Version]
  11. O’Connell, M.R. Molecular mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas systems. J. Mol. Biol. 2019, 431, 66–87. [Google Scholar] [CrossRef]
  12. Mohammadinejad, R.; Dehshahri, A.; Madamsetty, V.S.; Zahmatkeshan, M.; Tavakol, S.; Makvandi, P.; Khorsandi, D.; Pardakhty, A.; Ashrafizadeh, M.; Afshar, E.G. In vivo gene delivery mediated by non-viral vectors for cancer therapy. J. Control. Release 2020, 325, 249–275. [Google Scholar] [CrossRef]
  13. Biagioni, A.; Laurenzana, A.; Margheri, F.; Chillà, A.; Fibbi, G.; Del Rosso, M. Delivery systems of CRISPR/Cas9-based cancer gene therapy. J. Biol. Eng. 2018, 12, 1–9. [Google Scholar] [CrossRef] [Green Version]
  14. Fukuhara, H.; Ino, Y.; Todo, T. Oncolytic virus therapy: A new era of cancer treatment at dawn. Cancer Sci. 2016, 107, 1373–1379. [Google Scholar] [CrossRef]
  15. Chen, S.; Lee, B.; Lee, A.Y.-F.; Modzelewski, A.J.; He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 2016, 291, 14457–14467. [Google Scholar] [CrossRef] [Green Version]
  16. Kim, S.M.; Yang, Y.; Oh, S.J.; Hong, Y.; Seo, M.; Jang, M. Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting. J. Control. Release 2017, 266, 8–16. [Google Scholar] [CrossRef]
  17. Mout, R.; Ray, M.; Yesilbag Tonga, G.; Lee, Y.-W.; Tay, T.; Sasaki, K.; Rotello, V.M. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 2017, 11, 2452–2458. [Google Scholar] [CrossRef] [Green Version]
  18. Chen, C.; Ma, Y.; Du, S.; Wu, Y.; Shen, P.; Yan, T.; Li, X.; Song, Y.; Zha, Z.; Han, X. Controlled CRISPR-Cas9 Ribonucleoprotein Delivery for Sensitized Photothermal Therapy. Small 2021, 17, 2101155. [Google Scholar] [CrossRef]
  19. Taharabaru, T.; Yokoyama, R.; Higashi, T.; Mohammed, A.F.A.; Inoue, M.; Maeda, Y.; Niidome, T.; Onodera, R.; Motoyama, K. Genome editing in a wide area of the brain using dendrimer-based ternary polyplexes of Cas9 ribonucleoprotein. ACS Appl. Mater. Interfaces 2020, 12, 21386–21397. [Google Scholar] [CrossRef]
  20. Pan, X.; Pei, X.; Huang, H.; Su, N.; Wu, Z.; Wu, Z.; Qi, X. One-in-one individual package and delivery of CRISPR/Cas9 ribonucleoprotein using apoferritin. J. Control. Release 2021, 337, 686–697. [Google Scholar] [CrossRef]
  21. Wan, T.; Chen, Y.; Pan, Q.; Xu, X.; Kang, Y.; Gao, X.; Huang, F.; Wu, C.; Ping, Y. Genome editing of mutant KRAS through supramolecular polymer-mediated delivery of Cas9 ribonucleoprotein for colorectal cancer therapy. J. Control. Release 2020, 322, 236–247. [Google Scholar] [CrossRef]
  22. Sun, W.; Ji, W.; Hall, J.M.; Hu, Q.; Wang, C.; Beisel, C.L.; Gu, Z. Self-assembled DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing. Angew. Chem. 2015, 127, 12197–12201. [Google Scholar] [CrossRef]
  23. Dehshahri, A.; Oskuee, R.K.; Ramezani, M. Plasmid DNA delivery into hepatocytes using a multifunctional nanocarrier based on sugar-conjugated polyethylenimine. Gene. Mol. Biol. 2012, 14, 62–71. [Google Scholar]
  24. Sabahi, Z.; Samani, S.M.; Dehshahri, A. Conjugation of poly (amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin-12 gene. J. Biomater. Appl. 2015, 29, 941–953. [Google Scholar] [CrossRef]
  25. Khalvati, B.; Sheikhsaran, F.; Sharifzadeh, S.; Kalantari, T.; Behzad Behbahani, A.; Jamshidzadeh, A.; Dehshahri, A. Delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles. Artif. Cells Nanomed. Biotechnol. 2017, 45, 1036–1044. [Google Scholar] [CrossRef]
  26. Dehshahri, A.; Alhashemi, S.H.; Jamshidzadeh, A.; Sabahi, Z.; Samani, S.M.; Sadeghpour, H.; Mohazabieh, E.; Fadaei, M. Comparison of the effectiveness of polyethylenimine, polyamidoamine and chitosan in transferring plasmid encoding interleukin-12 gene into hepatocytes. Macromol. Res. 2013, 21, 1322–1330. [Google Scholar] [CrossRef]
  27. Sheikhsaran, F.; Sadeghpour, H.; Khalvati, B.; Entezar-Almahdi, E.; Dehshahri, A. Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene. Colloids Surf. B Biointerfaces 2017, 150, 426–436. [Google Scholar] [CrossRef] [Green Version]
  28. Nouri, F.; Sadeghpour, H.; Heidari, R.; Dehshahri, A. Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene. Int. J. Nanomed. 2017, 12, 5557. [Google Scholar] [CrossRef] [Green Version]
  29. Sadeghpour, H.; Khalvati, B.; Entezar-Almahdi, E.; Savadi, N.; Alhashemi, S.H.; Raoufi, M.; Dehshahri, A. Double domain polyethylenimine-based nanoparticles for integrin receptor mediated delivery of plasmid DNA. Sci. Rep. 2018, 8, 1–12. [Google Scholar] [CrossRef] [Green Version]
  30. Dehshahri, A.; Kazemi Oskuee, R.; Thomas Shier, W.; Ramezani, M. β-Galactosylated alkyl-oligoamine derivatives of polyethylenimine enhanced pDNA delivery into hepatic cells with reduced toxicity. Curr. Nanosci. 2012, 8, 548–555. [Google Scholar] [CrossRef]
  31. Dehshahri, A.; Ashrafizadeh, M.; Afshar, E.G.; Pardakhty, A.; Mandegary, A.; Mohammadinejad, R.; Sethi, G. Topoisomerase inhibitors: Pharmacology and emerging nanoscale delivery systems. Pharmacol. Res. 2020, 151, 104551. [Google Scholar] [CrossRef]
  32. Mohammadinejad, R.; Dehshahri, A.; Sassan, H.; Behnam, B.; Ashrafizadeh, M.; Samareh Gholami, A.; Pardakhty, A.; Mandegary, A. Preparation of carbon dot as a potential CRISPR/Cas9 plasmid delivery system for lung cancer cells. Minerva Biotecnol. 2020, 32, 106–113. [Google Scholar] [CrossRef]
  33. Ashrafizadeh, M.; Ahmadi, Z.; Kotla, N.G.; Afshar, E.G.; Samarghandian, S.; Mandegary, A.; Pardakhty, A.; Mohammadinejad, R.; Sethi, G. Nanoparticles targeting STATs in cancer therapy. Cells 2019, 8, 1158. [Google Scholar] [CrossRef] [Green Version]
  34. Zhang, S.; Shen, J.; Li, D.; Cheng, Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics 2021, 11, 614. [Google Scholar] [CrossRef]
  35. She, Z.-Y.; Yang, W.-X. SOX family transcription factors involved in diverse cellular events during development. Eur. J. Cell Biol. 2015, 94, 547–563. [Google Scholar] [CrossRef]
  36. Pagin, M.; Pernebrink, M.; Giubbolini, S.; Barone, C.; Sambruni, G.; Zhu, Y.; Chiara, M.; Ottolenghi, S.; Pavesi, G.; Wei, C.L. SOX2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network. Stem Cells 2021, 39, 1107–1119. [Google Scholar]
  37. Gross-Cohen, M.; Yanku, Y.; Kessler, O.; Barash, U.; Boyango, I.; Cid-Arregui, A.; Neufeld, G.; Ilan, N.; Vlodavsky, I. Heparanase 2 (Hpa2) attenuates tumor growth by inducing SOX2 expression. Matrix Biol. 2021, 99, 58–71. [Google Scholar] [CrossRef]
  38. Mehta, G.A.; Khanna, P.; Gatza, M.L. Emerging role of SOX proteins in breast Cancer development and maintenance. J. Mammary Gland Biol. Neoplasia 2019, 24, 213–230. [Google Scholar] [CrossRef]
  39. Nakagawa, M.; Koyanagi, M.; Tanabe, K.; Takahashi, K.; Ichisaka, T.; Aoi, T.; Okita, K.; Mochiduki, Y.; Takizawa, N.; Yamanaka, S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 2008, 26, 101–106. [Google Scholar] [CrossRef]
  40. Hou, L.; Srivastava, Y.; Jauch, R. Molecular basis for the genome engagement by Sox proteins. Semin. Cell Dev. Biol. 2017, 63, 2–12. [Google Scholar] [CrossRef] [Green Version]
  41. Schaefer, T.; Lengerke, C. SOX2 protein biochemistry in stemness, reprogramming, and cancer: The PI3K/AKT/SOX2 axis and beyond. Oncogene 2020, 39, 278–292. [Google Scholar] [CrossRef] [Green Version]
  42. Schock, E.N.; LaBonne, C. Sorting sox: Diverse roles for sox transcription factors during neural crest and craniofacial development. Front. Physiol. 2020, 11, 606889. [Google Scholar] [CrossRef]
  43. Williams, C.A.; Soufi, A.; Pollard, S.M. Post-translational modification of SOX family proteins: Key biochemical targets in cancer? Semin. CancerBiol. 2020, 67, 30–38. [Google Scholar] [CrossRef]
  44. Kumar, P.; Mistri, T.K. Transcription factors in SOX family: Potent regulators for cancer initiation and development in the human body. Semin. CancerBiol. 2020, 67, 105–113. [Google Scholar] [CrossRef]
  45. Abdelalim, E.M.; Emara, M.M.; Kolatkar, P.R. The SOX transcription factors as key players in pluripotent stem cells. Stem Cells Dev. 2014, 23, 2687–2699. [Google Scholar] [CrossRef]
  46. Grimm, D.; Bauer, J.; Wise, P.; Krüger, M.; Simonsen, U.; Wehland, M.; Infanger, M.; Corydon, T.J. The role of SOX family members in solid tumours and metastasis. Semin. Cancer Biol. 2020, 67, 122–153. [Google Scholar] [CrossRef]
  47. Buzzi, A.L.; Chen, J.; Thiery, A.; Delile, J.; Streit, A. Sox8 is Sufficient to Reprogram Ectoderm into Ear Vesicles and Associated Neurons. 2021. Available online: https://0-doi-org.brum.beds.ac.uk/10.1101/2021.04.20.440617 (accessed on 6 October 2021).
  48. Lopez-Caraballo, L.; Martorell-Marugan, J.; Carmona-Saez, P.; Gonzalez-Muñoz, E. Analysis of menstrual blood stromal cells reveals SOX15 triggers oocyte-based human cell reprogramming. Iscience 2020, 23, 101376. [Google Scholar] [CrossRef] [PubMed]
  49. Angelozzi, M.; Lefebvre, V. SOXopathies: Growing family of developmental disorders due to SOX mutations. Trends Genet. 2019, 35, 658–671. [Google Scholar] [CrossRef] [PubMed]
  50. Hoadley, K.A.; Yau, C.; Hinoue, T.; Wolf, D.M.; Lazar, A.J.; Drill, E.; Shen, R.; Taylor, A.M.; Cherniack, A.D.; Thorsson, V. Cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer. Cell 2018, 173, 291–304. e296. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Hori, N.; Yamane, M.; Kouno, K.; Sato, K. Induction of DNA demethylation depending on two sets of SOX2 and adjacent Oct3/4 binding sites (SOX-Oct motifs) within the mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region. J. Biol. Chem. 2012, 287, 44006–44016. [Google Scholar] [CrossRef] [Green Version]
  52. Kamachi, Y.; Uchikawa, M.; Tanouchi, A.; Sekido, R.; Kondoh, H. Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev. 2001, 15, 1272–1286. [Google Scholar] [CrossRef] [Green Version]
  53. Li, J.; Pan, G.; Cui, K.; Liu, Y.; Xu, S.; Pei, D. A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells. J. Biol. Chem. 2007, 282, 19481–19492. [Google Scholar] [CrossRef] [Green Version]
  54. Baltus, G.A.; Kowalski, M.P.; Zhai, H.; Tutter, A.V.; Quinn, D.; Wall, D.; Kadam, S. Acetylation of SOX2 induces its nuclear export in embryonic stem cells. Stem Cells 2009, 27, 2175–2184. [Google Scholar] [CrossRef] [PubMed]
  55. Yesudhas, D.; Anwar, M.A.; Panneerselvam, S.; Kim, H.K.; Choi, S. Evaluation of SOX2 binding affinities for distinct DNA patterns using steered molecular dynamics simulation. FEBS Open Bio 2017, 7, 1750–1767. [Google Scholar] [CrossRef]
  56. Wißmüller, S.; Kosian, T.; Wolf, M.; Finzsch, M.; Wegner, M. The high-mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors. Nucleic Acids Res. 2006, 34, 1735–1744. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Boyer, L.A.; Lee, T.I.; Cole, M.F.; Johnstone, S.E.; Levine, S.S.; Zucker, J.P.; Guenther, M.G.; Kumar, R.M.; Murray, H.L.; Jenner, R.G. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122, 947–956. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Knauss, J.L.; Miao, N.; Kim, S.-N.; Nie, Y.; Shi, Y.; Wu, T.; Pinto, H.B.; Donohoe, M.E.; Sun, T. Long noncoding RNA SOX2ot and transcription factor YY1 co-regulate the differentiation of cortical neural progenitors by repressing SOX2. Cell Death Dis. 2018, 9, 1–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Wang, Y.; Wu, N.; Luo, X.; Zhang, X.; Liao, Q.; Wang, J. SOX2OT, a novel tumor-related long non-coding RNA. Biomed. Pharmacother. 2020, 123, 109725. [Google Scholar] [CrossRef]
  60. Wang, Z.; Tan, M.; Chen, G.; Li, Z.; Lu, X. LncRNA SOX2-OT is a novel prognostic biomarker for osteosarcoma patients and regulates osteosarcoma cells proliferation and motility through modulating SOX2. IUBMB Life 2017, 69, 867–876. [Google Scholar] [CrossRef] [Green Version]
  61. Herrera-Solorio, A.M.; Peralta-Arrieta, I.; Armas López, L.; Hernández-Cigala, N.; Mendoza Milla, C.; Ortiz Quintero, B.; Catalan Cardenas, R.; Pineda Villegas, P.; Rodriguez Villanueva, E.; Trejo Iriarte, C.G. LncRNA SOX2-OT regulates AKT/ERK and SOX2/GLI-1 expression, hinders therapy, and worsens clinical prognosis in malignant lung diseases. Mol. Oncol. 2021, 15, 1110–1129. [Google Scholar] [CrossRef]
  62. Su, R.; Cao, S.; Ma, J.; Liu, Y.; Liu, X.; Zheng, J.; Chen, J.; Liu, L.; Cai, H.; Li, Z. Knockdown of SOX2OT inhibits the malignant biological behaviors of glioblastoma stem cells via up-regulating the expression of miR-194-5p and miR-122. Mol. Cancer 2017, 16, 1–22. [Google Scholar] [CrossRef] [PubMed]
  63. Wei, R.; Ding, C.; Rodrìguez, R.A.; Requena Mullor, M.d.M. The SOX2OT/miR-194-5p axis regulates cell proliferation and mobility of gastric cancer through suppressing epithelial-mesenchymal transition. Oncol. Lett. 2018, 16, 6361–6368. [Google Scholar] [CrossRef] [Green Version]
  64. Zhang, K.; Li, Y.; Qu, L.; Ma, X.; Zhao, H.; Tang, Y. Long noncoding RNA SOX2 overlapping transcript (SOX2OT) promotes non-small-cell lung cancer migration and invasion via sponging microRNA 132 (miR-132). OncoTargets Ther. 2018, 11, 5269. [Google Scholar]
  65. Liu, P.; Chen, M.; Liu, Y.; Qi, L.S.; Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 2018, 22, 252–261. e254. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Miyagi, S.; Saito, T.; Mizutani, K.-i.; Masuyama, N.; Gotoh, Y.; Iwama, A.; Nakauchi, H.; Masui, S.; Niwa, H.; Nishimoto, M. The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells. Mol. Cell. Biol. 2004, 24, 4207–4220. [Google Scholar] [CrossRef] [Green Version]
  67. Zhou, H.Y.; Katsman, Y.; Dhaliwal, N.K.; Davidson, S.; Macpherson, N.N.; Sakthidevi, M.; Collura, F.; Mitchell, J.A. A SOX2 distal enhancer cluster regulates embryonic stem cell differentiation potential. Genes Dev. 2014, 28, 2699–2711. [Google Scholar] [CrossRef] [Green Version]
  68. Li, Y.; Rivera, C.M.; Ishii, H.; Jin, F.; Selvaraj, S.; Lee, A.Y.; Dixon, J.R.; Ren, B. CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells. PLoS ONE 2014, 9, e114485. [Google Scholar] [CrossRef] [Green Version]
  69. de Wit, E.; Vos, E.S.; Holwerda, S.J.; Valdes-Quezada, C.; Verstegen, M.J.; Teunissen, H.; Splinter, E.; Wijchers, P.J.; Krijger, P.H.; de Laat, W. CTCF binding polarity determines chromatin looping. Mol. Cell 2015, 60, 676–684. [Google Scholar] [CrossRef] [Green Version]
  70. Takahashi, K.; Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006, 126, 663–676. [Google Scholar] [CrossRef] [Green Version]
  71. Avilion, A.A.; Nicolis, S.K.; Pevny, L.H.; Perez, L.; Vivian, N.; Lovell-Badge, R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003, 17, 126–140. [Google Scholar] [CrossRef] [Green Version]
  72. Zhao, S.; Nichols, J.; Smith, A.G.; Li, M. SoxB transcription factors specify neuroectodermal lineage choice in ES cells. Mol. Cell. Neurosci. 2004, 27, 332–342. [Google Scholar] [CrossRef] [PubMed]
  73. Que, J.; Okubo, T.; Goldenring, J.R.; Nam, K.-T.; Kurotani, R.; Morrisey, E.E.; Taranova, O.; Pevny, L.H.; Hogan, B.L. Multiple dose-dependent roles for SOX2 in the patterning and differentiation of anterior foregut endoderm. Development 2007, 134, 2521–2531. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Andreucci, E.; Pietrobono, S.; Peppicelli, S.; Ruzzolini, J.; Bianchini, F.; Biagioni, A.; Stecca, B.; Calorini, L. SOX2 as a novel contributor of oxidative metabolism in melanoma cells. Cell Commun. Signal. 2018, 16, 1–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Barone, C.; Buccarelli, M.; Alessandrini, F.; Pagin, M.; Rigoldi, L.; Sambruni, I.; Favaro, R.; Ottolenghi, S.; Pallini, R.; Ricci-Vitiani, L. SOX2-dependent maintenance of mouse oligodendroglioma involves the SOX2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2. Glia 2021, 69, 579–593. [Google Scholar] [CrossRef] [PubMed]
  76. Zhang, S.; Xiong, X.; Sun, Y. Functional characterization of SOX2 as an anticancer target. Signal. Transduct. Target. Ther. 2020, 5, 1–17. [Google Scholar] [CrossRef]
  77. Bareiss, P.M.; Paczulla, A.; Wang, H.; Schairer, R.; Wiehr, S.; Kohlhofer, U.; Rothfuss, O.C.; Fischer, A.; Perner, S.; Staebler, A. SOX2 expression associates with stem cell state in human ovarian carcinoma. Cancer Res. 2013, 73, 5544–5555. [Google Scholar] [CrossRef] [Green Version]
  78. Lengerke, C.; Fehm, T.; Kurth, R.; Neubauer, H.; Scheble, V.; Müller, F.; Schneider, F.; Petersen, K.; Wallwiener, D.; Kanz, L. Expression of the embryonic stem cell marker SOX2 in early-stage breast carcinoma. BMC Cancer 2011, 11, 42. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Mansouri, S.; Nejad, R.; Karabork, M.; Ekinci, C.; Solaroglu, I.; Aldape, K.D.; Zadeh, G. SOX2: Regulation of expression and contribution to brain tumors. CNS Oncol. 2016, 5, 159–173. [Google Scholar] [CrossRef]
  80. Wang, S.; Tie, J.; Wang, R.; Hu, F.; Gao, L.; Wang, W.; Wang, L.; Li, Z.; Hu, S.; Tang, S. SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN. Cancer Lett. 2015, 358, 210–219. [Google Scholar] [CrossRef]
  81. Mou, W.; Xu, Y.; Ye, Y.; Chen, S.; Li, X.; Gong, K.; Liu, Y.; Chen, Y.; Li, X.; Tian, Y. Expression of Sox2 in breast cancer cells promotes the recruitment of M2 macrophages to tumor microenvironment. Cancer Lett. 2015, 358, 115–123. [Google Scholar] [CrossRef]
  82. Mukherjee, P.; Gupta, A.; Chattopadhyay, D.; Chatterji, U. Modulation of SOX2 expression delineates an end-point for paclitaxel-effectiveness in breast cancer stem cells. Sci. Rep. 2017, 7, 1–16. [Google Scholar] [CrossRef] [PubMed]
  83. Hüser, L.; Sachindra, S.; Granados, K.; Federico, A.; Larribère, L.; Novak, D.; Umansky, V.; Altevogt, P.; Utikal, J. SOX2-mediated upregulation of CD24 promotes adaptive resistance toward targeted therapy in melanoma. Int. J. Cancer 2018, 143, 3131–3142. [Google Scholar] [CrossRef] [Green Version]
  84. Song, W.-S.; Yang, Y.-P.; Huang, C.-S.; Lu, K.-H.; Liu, W.-H.; Wu, W.-W.; Lee, Y.-Y.; Lo, W.-L.; Lee, S.-D.; Chen, Y.-W. SOX2, a stemness gene, regulates tumor-initiating and drug-resistant properties in CD133-positive glioblastoma stem cells. J. Chin. Med Assoc. 2016, 79, 538–545. [Google Scholar] [CrossRef] [Green Version]
  85. Saenz-Antoñanzas, A.; Moncho-Amor, V.; Auzmendi-Iriarte, J.; Elua-Pinin, A.; Rizzoti, K.; Lovell-Badge, R.; Matheu, A. CRISPR/Cas9 Deletion of SOX2 Regulatory Region 2 (SRR2) Decreases SOX2 Malignant Activity in Glioblastoma. Cancers 2021, 13, 1574. [Google Scholar] [CrossRef]
  86. Li, P.-Y.; Li, S.-Q.; Gao, S.-G.; Dong, D.-Y. CRISPR/Cas9-mediated gene editing on SOX2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development in mice. Biochem. Biophys. Res. Commun. 2021, 573, 107–111. [Google Scholar] [CrossRef]
  87. Dong, D.-Y.; Li, P.-Y. Identifying SOX2-OT transcript that is responsible for regulating SOX2 in cancer cells and embryonic stem cells. Res. Ideas Outcomes 2021, 7, e69726. [Google Scholar] [CrossRef]
  88. Yang, S.C.; Liu, J.J.; Wang, C.K.; Lin, Y.T.; Tsai, S.Y.; Chen, W.J.; Huang, W.K.; Tu, P.W.A.; Lin, Y.C.; Chang, C.F. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2. FASEB J. 2019, 33, 10577–10592. [Google Scholar] [CrossRef] [Green Version]
  89. Cheng, M.; Jin, X.; Mu, L.; Wang, F.; Li, W.; Zhong, X.; Liu, X.; Shen, W.; Liu, Y.; Zhou, Y. Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development. J. Neurosci. Res. 2016, 94, 814–824. [Google Scholar] [CrossRef]
  90. Panaliappan, T.K.; Wittmann, W.; Jidigam, V.K.; Mercurio, S.; Bertolini, J.A.; Sghari, S.; Bose, R.; Patthey, C.; Nicolis, S.K.; Gunhaga, L. SOX2 is required for olfactory pit formation and olfactory neurogenesis through BMP restriction and Hes5 upregulation. Development 2018, 145, 153791. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  91. Schaefer, S.M.; Segalada, C.; Cheng, P.; Bonalli, M.; Parfejevs, V.; Levesque, M.P.; Dummer, R.; Nicolis, S.K.; Sommer, L. SOX2 is dispensable for primary melanoma and metastasis formation. Oncogene 2017, 36, 4516–4524. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Menon, D.R.; Luo, Y.; Arcaroli, J.J.; Liu, S.; KrishnanKutty, L.N.; Osborne, D.G.; Li, Y.; Samson, J.M.; Bagby, S.; Tan, A.-C. CDK1 interacts with SOX2 and promotes tumor initiation in human melanoma. Cancer Res. 2018, 78, 6561–6574. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Maurizi, G.; Verma, N.; Gadi, A.; Mansukhani, A.; Basilico, C. SOX2 is required for tumor development and cancer cell proliferation in osteosarcoma. Oncogene 2018, 37, 4626–4632. [Google Scholar] [CrossRef] [PubMed]
  94. Nettersheim, D.; Vadder, S.; Jostes, S.; Heimsoeth, A.; Schorle, H. TCam-2 cells deficient for SOX2 and FOXA2 are blocked in differentiation and maintain a seminoma-like cell fate in vivo. Cancers 2019, 11, 728. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  95. Balboa, D.; Weltner, J.; Novik, Y.; Eurola, S.; Wartiovaara, K.; Otonkoski, T. Generation of a SOX2 reporter human induced pluripotent stem cell line using CRISPR/SaCas9. Stem Cell Res. 2017, 22, 16–19. [Google Scholar] [CrossRef] [PubMed]
  96. Bressan, R.B.; Dewari, P.S.; Kalantzaki, M.; Gangoso, E.; Matjusaitis, M.; Garcia-Diaz, C.; Blin, C.; Grant, V.; Bulstrode, H.; Gogolok, S. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells. Development 2017, 144, 635–648. [Google Scholar] [CrossRef] [Green Version]
  97. Yang, H.; Wang, H.; Shivalila, C.S.; Cheng, A.W.; Shi, L.; Jaenisch, R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 2013, 154, 1370–1379. [Google Scholar] [CrossRef] [Green Version]
  98. Mei, Y.; Cai, D.; Dai, X. Modulating cancer stemness provides luminal a breast cancer cells with HER2 positive-like features. J. Cancer 2020, 11, 1162. [Google Scholar] [CrossRef] [Green Version]
  99. Chang, Y.K.; Hwang, J.S.; Chung, T.Y.; Shin, Y.J. SOX2 activation using CRISPR/dCas9 promotes wound healing in corneal endothelial cells. Stem Cells 2018, 36, 1851–1862. [Google Scholar] [CrossRef] [Green Version]
  100. Fei, J.-F.; Schuez, M.; Tazaki, A.; Taniguchi, Y.; Roensch, K.; Tanaka, E.M. CRISPR-mediated genomic deletion of SOX2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem Cell Rep. 2014, 3, 444–459. [Google Scholar] [CrossRef] [Green Version]
  101. Wu, Y.; Chang, T.; Long, Y.; Huang, H.; Kandeel, F.; Yee, J.-K. Using gene editing to establish a safeguard system for pluripotent stem-cell-based therapies. Iscience 2019, 22, 409–422. [Google Scholar] [CrossRef] [Green Version]
  102. Chen, Y.; Cao, J.; Xiong, M.; Petersen, A.J.; Dong, Y.; Tao, Y.; Huang, C.T.-L.; Du, Z.; Zhang, S.-C. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 2015, 17, 233–244. [Google Scholar] [CrossRef] [Green Version]
  103. Haas, B.J.; Whited, J.L. Advances in decoding axolotl limb regeneration. Trends Genet. 2017, 33, 553–565. [Google Scholar] [CrossRef]
  104. Fei, J.-F.; Schuez, M.; Knapp, D.; Taniguchi, Y.; Drechsel, D.N.; Tanaka, E.M. Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. Proc. Natl. Acad. Sci. USA 2017, 114, 12501–12506. [Google Scholar] [CrossRef] [Green Version]
  105. Fei, J.-F.; Knapp, D.; Schuez, M.; Murawala, P.; Zou, Y.; Singh, S.P.; Drechsel, D.; Tanaka, E.M. Tissue-and time-directed electroporation of CAS9 protein–gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration. NPJ Regen. Med. 2016, 1, 1–9. [Google Scholar] [CrossRef] [Green Version]
  106. Blank, A.; Dekker, C.A. Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Biochemistry 1981, 20, 2261–2267. [Google Scholar] [CrossRef]
  107. Pritchett, J.; Athwal, V.; Roberts, N.; Hanley, N.A.; Hanley, K.P. Understanding the role of SOX9 in acquired diseases: Lessons from development. Trends Mol. Med. 2011, 17, 166–174. [Google Scholar] [CrossRef]
  108. Jo, A.; Denduluri, S.; Zhang, B.; Wang, Z.; Yin, L.; Yan, Z.; Kang, R.; Shi, L.L.; Mok, J.; Lee, M.J. The versatile functions of Sox9 in development, stem cells, and human diseases. Genes Dis. 2014, 1, 149–161. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  109. Fonseca, A.C.S.; Bonaldi, A.; Bertola, D.R.; Kim, C.A.; Otto, P.A.; Vianna-Morgante, A.M. The clinical impact of chromosomal rearrangements with breakpoints upstream of the SOX9gene: Two novel de novo balanced translocations associated with acampomelic campomelic dysplasia. BMC Med Genet. 2013, 14, 50. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  110. Foster, J.W.; Dominguez-Steglich, M.A.; Guioli, S.; Kwok, C.; Weller, P.A.; Stevanović, M.; Weissenbach, J.; Mansour, S.; Young, I.D.; Goodfellow, P.N. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 1994, 372, 525–530. [Google Scholar] [CrossRef]
  111. Wagner, T.; Wirth, J.; Meyer, J.; Zabel, B.; Held, M.; Zimmer, J.; Pasantes, J.; Bricarelli, F.D.; Keutel, J.; Hustert, E. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 1994, 79, 1111–1120. [Google Scholar] [CrossRef]
  112. Kashimada, K.; Koopman, P. Sry: The master switch in mammalian sex determination. Development 2010, 137, 3921–3930. [Google Scholar] [CrossRef] [Green Version]
  113. Sekido, R.; Lovell-Badge, R. Sex determination and SRY: Down to a wink and a nudge? Trends Genet. 2009, 25, 19–29. [Google Scholar] [CrossRef]
  114. Sekido, R.; Lovell-Badge, R. Sex determination involves synergistic action of SRY and SF1 on a specific SOX9 enhancer. Nature 2008, 453, 930–934. [Google Scholar] [CrossRef] [PubMed]
  115. Gonen, N.; Quinn, A.; O’Neill, H.C.; Koopman, P.; Lovell-Badge, R. Normal levels of SOX9 expression in the developing mouse testis depend on the TES/TESCO enhancer, but this does not act alone. PLoS Genet. 2017, 13, e1006520. [Google Scholar]
  116. Ogawa, Y.; Terao, M.; Hara, S.; Tamano, M.; Okayasu, H.; Kato, T.; Takada, S. Mapping of a responsible region for sex reversal upstream of SOX9 by production of mice with serial deletion in a genomic locus. Sci. Rep. 2018, 8, 1–10. [Google Scholar] [CrossRef] [PubMed]
  117. Hong, Y.; Chen, W.; Du, X.; Ning, H.; Chen, H.; Shi, R.; Lin, S.; Xu, R.; Zhu, J.; Wu, S. Upregulation of sex-determining region Y-box 9 (SOX9) promotes cell proliferation and tumorigenicity in esophageal squamous cell carcinoma. Oncotarget 2015, 6, 31241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  118. Song, S.; Maru, D.M.; Ajani, J.A.; Chan, C.-H.; Honjo, S.; Lin, H.-K.; Correa, A.; Hofstetter, W.L.; Davila, M.; Stroehlein, J. Loss of TGF-β adaptor β2SP activates notch signaling and SOX9 expression in esophageal adenocarcinoma. Cancer Res. 2013, 73, 2159–2169. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  119. Song, S.; Ajani, J.A.; Honjo, S.; Maru, D.M.; Chen, Q.; Scott, A.W.; Heallen, T.R.; Xiao, L.; Hofstetter, W.L.; Weston, B. Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties. Cancer Res. 2014, 74, 4170–4182. [Google Scholar] [CrossRef] [Green Version]
  120. Ramachandran, L.; Manu, K.A.; Shanmugam, M.K.; Li, F.; Siveen, K.S.; Vali, S.; Kapoor, S.; Abbasi, T.; Surana, R.; Smoot, D.T.; et al. Isorhamnetin inhibits proliferation and invasion and induces apoptosis through the modulation of peroxisome proliferator-activated receptor gamma activation pathway in gastric cancer. J. Biol. Chem. 2012, 287, 38028–38040. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  121. Song, S.; Wang, Z.; Li, Y.; Ma, L.; Jin, J.; Scott, A.W.; Xu, Y.; Estrella, J.S.; Song, Y.; Liu, B. PPARδ Interacts with the Hippo Coactivator YAP1 to Promote SOX9 Expression and Gastric Cancer Progression. Mol. Cancer Res. 2020, 18, 390–402. [Google Scholar] [CrossRef]
  122. Malhotra, G.K.; Zhao, X.; Edwards, E.; Kopp, J.L.; Naramura, M.; Sander, M.; Band, H.; Band, V. The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells. BMC Dev. Biol. 2014, 14, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  123. Chakravarty, G.; Moroz, K.; Makridakis, N.M.; Lloyd, S.A.; Galvez, S.E.; Canavello, P.R.; Lacey, M.R.; Agrawal, K.; Mondal, D. Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer. Exp. Biol. Med. 2011, 236, 145–155. [Google Scholar] [CrossRef]
  124. Domenici, G.; Aurrekoetxea-Rodríguez, I.; Simões, B.M.; Rábano, M.; Lee, S.Y.; San Millán, J.; Comaills, V.; Oliemuller, E.; López-Ruiz, J.A.; Zabalza, I. A SOX2–SOX9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells. Oncogene 2019, 38, 3151–3169. [Google Scholar] [CrossRef] [PubMed]
  125. Li, X.; Tao, Y.; Bradley, R.; Du, Z.; Tao, Y.; Kong, L.; Dong, Y.; Jones, J.; Yan, Y.; Harder, C.R. Fast generation of functional subtype astrocytes from human pluripotent stem cells. Stem Cell Rep. 2018, 11, 998–1008. [Google Scholar] [CrossRef] [PubMed]
  126. Ran, F.A.; Hsu, P.D.; Wright, J.; Agarwala, V.; Scott, D.A.; Zhang, F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013, 8, 2281–2308. [Google Scholar] [CrossRef] [Green Version]
  127. Liu, Z.; Yao, M.; Yao, H.; Hu, G.; Qin, B. Generation of Rybp homozygous knockout murine ES cell line GIBHe001-A-1 by using CRISPR/Cas9 technology. Stem Cell Res. 2019, 41, 101638. [Google Scholar] [CrossRef]
  128. Adikusuma, F.; Pederick, D.; McAninch, D.; Hughes, J.; Thomas, P. Functional equivalence of the SOX2 and SOX3 transcription factors in the developing mouse brain and testes. Genetics 2017, 206, 1495–1503. [Google Scholar] [CrossRef] [Green Version]
  129. Gou, Y.; Guo, J.; Maulding, K.; Riley, B.B. SOX2 and SOX3 cooperate to regulate otic/epibranchial placode induction in zebrafish. Dev. Biol. 2018, 435, 84–95. [Google Scholar] [CrossRef]
  130. Hong, Q.; Li, C.; Ying, R.; Lin, H.; Li, J.; Zhao, Y.; Cheng, H.; Zhou, R. Loss-of-function of SOX3 causes follicle development retardation and reduces fecundity in zebrafish. Protein Cell 2019, 10, 347–364. [Google Scholar] [CrossRef] [Green Version]
  131. Moran, J.D.; Kim, H.H.; Li, Z.; Moreno, C.S. SOX4 regulates invasion of bladder cancer cells via repression of WNT5a. Int. J. Oncol. 2019, 55, 359–370. [Google Scholar] [CrossRef] [Green Version]
  132. Cantù, C.; Ierardi, R.; Alborelli, I.; Fugazza, C.; Cassinelli, L.; Piconese, S.; Bosè, F.; Ottolenghi, S.; Ferrari, G.; Ronchi, A. SOX6 enhances erythroid differentiation in human erythroid progenitors. Blood 2011, 117, 3669–3679. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  133. Banan, M.; Bayat, H.; Azarkeivan, A.; Mohammadparast, S.; Kamali, K.; Farashi, S.; Bayat, N.; Khani, M.H.; Neishabury, M.; Najmabadi, H. The X mn I and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian β-thalassemia patients. Hemoglobin 2012, 36, 371–380. [Google Scholar] [CrossRef] [PubMed]
  134. Banan, M.; Bayat, H.; Namdar-Aligoodarzi, P.; Azarkeivan, A.; Kamali, K.; Daneshmand, P.; Zaker-Kandjani, B.; Najmabadi, H. Utility of the multivariate approach in predicting β-thalassemia intermedia or β-thalassemia major types in Iranian patients. Hemoglobin 2013, 37, 413–422. [Google Scholar] [CrossRef] [PubMed]
  135. Shariati, L.; Rohani, F.; Heidari Hafshejani, N.; Kouhpayeh, S.; Boshtam, M.; Mirian, M.; Rahimmanesh, I.; Hejazi, Z.; Modarres, M.; Pieper, I.L. Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of β-thalassemia. J. Cell. Biochem. 2018, 119, 9357–9363. [Google Scholar] [CrossRef]
  136. Bondurand, N.; Sham, M.H. The role of SOX10 during enteric nervous system development. Dev. Biol. 2013, 382, 330–343. [Google Scholar] [CrossRef] [Green Version]
  137. Elmaleh-Berges, M.; Baumann, C.; Noël-Pétroff, N.; Sekkal, A.; Couloigner, V.; Devriendt, K.; Wilson, M.; Marlin, S.; Sebag, G.; Pingault, V. Spectrum of temporal bone abnormalities in patients with Waardenburg syndrome and SOX10 mutations. Am. J. Neuroradiol. 2013, 34, 1257–1263. [Google Scholar] [CrossRef] [Green Version]
  138. Chaoui, A.; Watanabe, Y.; Touraine, R.; Baral, V.; Goossens, M.; Pingault, V.; Bondurand, N. Identification and functional analysis of SOX10 missense mutations in different subtypes of Waardenburg syndrome. Hum. Mutat. 2011, 32, 1436–1449. [Google Scholar] [CrossRef] [Green Version]
  139. Zhou, X.; Wang, L.; Du, Y.; Xie, F.; Li, L.; Liu, Y.; Liu, C.; Wang, S.; Zhang, S.; Huang, X. Efficient generation of gene-modified pigs harboring precise orthologous human mutation via CRISPR/Cas9-induced homology-directed repair in zygotes. Hum. Mutat. 2016, 37, 110–118. [Google Scholar] [CrossRef]
  140. Gandhi, S.; Piacentino, M.L.; Vieceli, F.M.; Bronner, M.E. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo. Dev. Biol. 2017, 432, 86–97. [Google Scholar] [CrossRef]
  141. Horikiri, T.; Ohi, H.; Shibata, M.; Ikeya, M.; Ueno, M.; Sotozono, C.; Kinoshita, S.; Sato, T. SOX10-nano-lantern reporter human iPS cells; a versatile tool for neural crest research. PLoS ONE 2017, 12, e0170342. [Google Scholar] [CrossRef]
  142. Simon, C.; Lickert, H.; Götz, M.; Dimou, L. Sox10-iCreERT2: A mouse line to inducibly trace the neural crest and oligodendrocyte lineage. Genesis 2012, 50, 506–515. [Google Scholar] [CrossRef] [PubMed]
  143. Iannaccone, P.; Jacob, H. Rats! Dis. Model. Mech. 2009, 2, 206–210. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  144. Chen, Y.; Spitzer, S.; Agathou, S.; Karadottir, R.T.; Smith, A. Gene editing in rat embryonic stem cells to produce in vitro models and in vivo reporters. Stem Cell Rep. 2017, 9, 1262–1274. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  145. Matjusaitis, M.; Wagstaff, L.J.; Martella, A.; Baranowski, B.; Blin, C.; Gogolok, S.; Williams, A.; Pollard, S.M. Reprogramming of fibroblasts to oligodendrocyte progenitor-like cells using CRISPR/Cas9-based synthetic transcription factors. Stem Cell Rep. 2019, 13, 1053–1067. [Google Scholar] [CrossRef] [Green Version]
  146. Jankowski, M.P.; Cornuet, P.K.; McIlwrath, S.; Koerber, H.R.; Albers, K.M. SRY-box containing gene 11 (Sox11) transcription factor is required for neuron survival and neurite growth. Neuroscience 2006, 143, 501–514. [Google Scholar] [CrossRef] [Green Version]
  147. Perry, R.B.-T.; Hezroni, H.; Goldrich, M.J.; Ulitsky, I. Regulation of neuroregeneration by long noncoding RNAs. Mol. Cell 2018, 72, 553–567. [Google Scholar] [CrossRef] [Green Version]
  148. Patodia, S.; Raivich, G. Role of transcription factors in peripheral nerve regeneration. Front. Mol. Neurosci. 2012, 5, 8. [Google Scholar] [CrossRef] [Green Version]
  149. Suzuki, H.; DinH, T.T.H.; DaiToku, Y.; TanimoTo, Y.; Kato, K.; Azami, T.; Ema, M.; Murata, K.; Mizuno, S.; Sugiyama, F. Generation of bicistronic reporter knockin mice for visualizing germ layers. Exp. Anim. 2019, 68, 499–509. [Google Scholar] [CrossRef]
  150. Guimarães-Young, A.; Neff, T.; Dupuy, A.J.; Goodheart, M.J. Conditional deletion of SOX17 reveals complex effects on uterine adenogenesis and function. Dev. Biol. 2016, 414, 219–227. [Google Scholar] [CrossRef]
  151. Wang, X.; Li, X.; Wang, T.; Wu, S.-P.; Jeong, J.-W.; Kim, T.H.; Young, S.L.; Lessey, B.A.; Lanz, R.B.; Lydon, J.P. SOX17 regulates uterine epithelial–stromal cross-talk acting via a distal enhancer upstream of Ihh. Nat. Commun. 2018, 9, 1–14. [Google Scholar] [CrossRef]
  152. Kimura, Y.; Oda, M.; Nakatani, T.; Sekita, Y.; Monfort, A.; Wutz, A.; Mochizuki, H.; Nakano, T. CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells. Sci. Rep. 2015, 5, 10710. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  153. Nagao, Y.; Takada, H.; Miyadai, M.; Adachi, T.; Seki, R.; Kamei, Y.; Hara, I.; Taniguchi, Y.; Naruse, K.; Hibi, M. Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish. PLoS Genet. 2018, 14, e1007260. [Google Scholar] [CrossRef] [PubMed]
  154. Li, L.; Feng, J.; Zhao, S.; Rong, Z.; Lin, Y. SOX9 inactivation affects the proliferation and differentiation of human lung organoids. Stem Cell Res. Ther. 2021, 12, 1–12. [Google Scholar] [CrossRef] [PubMed]
  155. Turan, S.; Boerstler, T.; Kavyanifar, A.; Loskarn, S.; Reis, A.; Winner, B.; Lie, D.C. A novel human stem cell model for Coffin–Siris syndrome-like syndrome reveals the importance of SOX11 dosage for neuronal differentiation and survival. Hum. Mol. Genet. 2019, 28, 2589–2599. [Google Scholar] [CrossRef]
  156. Frangoul, H.; Altshuler, D.; Cappellini, M.D.; Chen, Y.-S.; Domm, J.; Eustace, B.K.; Foell, J.; de la Fuente, J.; Grupp, S.; Handgretinger, R. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 2021, 384, 252–260. [Google Scholar] [CrossRef] [PubMed]
Figure 1. CRISPR-Cas9 mediated gene editing. A single Cas9 effector recognizes the target region and blunt-ended double-stranded breaks (DSBs) would be induced at target site through Cas9 endonuclease domains. The DSBs introduced by Cas9 endonuclease would promptly be repaired by the error-prone NHEJ pathway or by most specific HDR. The NHEJ pathway might result in random Indels and disrupt the sequence frame at the target site. Alternatively, when a repair template is supplied, the HDR pathway increases the accuracy and efficiency of the targeted gene editing.
Figure 1. CRISPR-Cas9 mediated gene editing. A single Cas9 effector recognizes the target region and blunt-ended double-stranded breaks (DSBs) would be induced at target site through Cas9 endonuclease domains. The DSBs introduced by Cas9 endonuclease would promptly be repaired by the error-prone NHEJ pathway or by most specific HDR. The NHEJ pathway might result in random Indels and disrupt the sequence frame at the target site. Alternatively, when a repair template is supplied, the HDR pathway increases the accuracy and efficiency of the targeted gene editing.
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Figure 2. Subfamiles of SOX proteins and the functional domains. The major protein functional domains include the high-mobility group (HMG) domain, transactivation domain (TAD), coiled-coil (CC) domain, transrepression domain (TRD), dimerization (DIM) domain, and SoxB homology domain. Reprinted by permission from Frontiers, Frontiers in Physiology [42], Copyright 2020.
Figure 2. Subfamiles of SOX proteins and the functional domains. The major protein functional domains include the high-mobility group (HMG) domain, transactivation domain (TAD), coiled-coil (CC) domain, transrepression domain (TRD), dimerization (DIM) domain, and SoxB homology domain. Reprinted by permission from Frontiers, Frontiers in Physiology [42], Copyright 2020.
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Figure 3. Developmental aspects of SOX protein subfamilies. SOX genes play crucial functions in hair, skin, eye, auditive system, musculoskeletal system, cardiovascular system, nervous system, gastrointestinal system, reproductive system, and embryonic development. Reprinted by permission from Elsevier, Trends in Genetics [49], Copyright 2019.
Figure 3. Developmental aspects of SOX protein subfamilies. SOX genes play crucial functions in hair, skin, eye, auditive system, musculoskeletal system, cardiovascular system, nervous system, gastrointestinal system, reproductive system, and embryonic development. Reprinted by permission from Elsevier, Trends in Genetics [49], Copyright 2019.
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Figure 4. The involvement of SOX protein subfamiles in human cancers. SOX genes can be upregulated or downregulated in tumor cells including skin cancer, bone cancer, brain cancers, respiratory cancers, gastrointestinal cancers, leukemia, urological cancers, gynecologic cancers, and breast cancer. Reprinted by permission from Elsevier, Seminars in Cancer Biology [46], Copyright 2020.
Figure 4. The involvement of SOX protein subfamiles in human cancers. SOX genes can be upregulated or downregulated in tumor cells including skin cancer, bone cancer, brain cancers, respiratory cancers, gastrointestinal cancers, leukemia, urological cancers, gynecologic cancers, and breast cancer. Reprinted by permission from Elsevier, Seminars in Cancer Biology [46], Copyright 2020.
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Figure 5. SOX2 cross-talks with various cellular pathways. SOX2 cross-talks with some signaling pathways including TGF-β, WNT, HIPPO, Hedgehog, and EGFR regulates cell proliferation, survival, migration, and differentiation. Reprinted by permission from Springer Nature, Signal Transduction and Targeted Therapy [76], Copyright 2020.
Figure 5. SOX2 cross-talks with various cellular pathways. SOX2 cross-talks with some signaling pathways including TGF-β, WNT, HIPPO, Hedgehog, and EGFR regulates cell proliferation, survival, migration, and differentiation. Reprinted by permission from Springer Nature, Signal Transduction and Targeted Therapy [76], Copyright 2020.
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Figure 6. Applications of the CRISPR system to edit SOX2. Editing SOX2 by CRISPR-Cas affects cell viability, proliferation, pluripotency, and tissue regeneration.
Figure 6. Applications of the CRISPR system to edit SOX2. Editing SOX2 by CRISPR-Cas affects cell viability, proliferation, pluripotency, and tissue regeneration.
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Table 1. CRISPR targeting SOX2.
Table 1. CRISPR targeting SOX2.
Cell Line/Animal NameKO/KIOutcome Refs
TCam-2 Cell lineKOMaintaining a seminoma-cell fate in vivo for about six weeks[94]
Human corneal endothelial cells (hCECs)Activation systemRegenerating hCECs[99]
MouseKOAbolishing tumorigenicity and suppressing CSC phenotype[93]
iPSCsKOInducing pluripotency [65]
Human melanoma cellsKODemonstrating loss of SOX2 did neither affect melanoma initiation and growth, nor metastasis formation.[91]
AxolotlKOShowing loss of neural stem cell amplification during axolotl tail
regeneration
[100]
ESCsKIIntegrating a suicide gene in-frame to end SOX2 to inhibit
differentiation
[101]
ESCsKOEstablishing a method for conditional KO by using CRISPR-Cas9[102]
U373MGDeletionDeletion of SOX2 regulatory region 2 (SRR2) reduces SOX2 expression, halts malignant activity of SOX2, and impairs tumor initiation and progression[85]
NSCsKIMonitoring the expression rate of SOX2 gene[96]
HEL24.3KIGenerating hiPSC which contains a SOX2-ntdTomato reporter,
to study the expression of SOX2 in live cells.
[95]
Table 2. CRISPR targeting other SOX genes.
Table 2. CRISPR targeting other SOX genes.
Gene NameCell Line/
Animal Name
Knock Out/Knock InOutcomeRefs
SOX1ESCsKIFor engineering the haploid ES cell genome[152]
SOX2, SOX3ZebrafishKOSOX2 and SOX3 are important for the normal
development of otic and epibranchial placodes
[129]
SOX2, SOX3MiceGene-swap (KO/KI)SOX2 and SOX3 proteins are functionally equivalent in brain and testes[128]
SOX3ZebrafishKOSOX3 is important for follicle development and
fecundity in zebrafish
[127]
SOX4The bladder cancer cell linesKnockdownDecreasing invasive capabilities in bladder cancer[131]
SOX5, SOX10Medaka, zebrafishKODemonstrating interaction between SOX5 and SOX10[153]
SOX6K562 cell lineKOLeading to γ-globin reactivation[135]
SOX9H9 hESC lineKOAffecting human lung organoids proliferation and differentiation[154]
SOX10NSCsActivation systemEnhancing neural stem cell differentiation[145]
SOX10Chicken fibroblast
cell line
KOOptimizing of genome editing approach in early chick embryos and perturbing downstream neural crest GRN components[140]
SOX10hiPSCKIGenerating neural crest progenitor cells by adding a reporter gene into SOX10 locus[141]
SOX11SCsKOHaplo insufficiency of SOX11 impairs key processes of human neurodevelopment[155]
SOX17MiceKOSOX17 is critical for embryo implantation and
pregnancy
[151]
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Dehshahri, A.; Biagioni, A.; Bayat, H.; Lee, E.H.C.; Hashemabadi, M.; Fekri, H.S.; Zarrabi, A.; Mohammadinejad, R.; Kumar, A.P. Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives. Int. J. Mol. Sci. 2021, 22, 11321. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111321

AMA Style

Dehshahri A, Biagioni A, Bayat H, Lee EHC, Hashemabadi M, Fekri HS, Zarrabi A, Mohammadinejad R, Kumar AP. Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives. International Journal of Molecular Sciences. 2021; 22(21):11321. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111321

Chicago/Turabian Style

Dehshahri, Ali, Alessio Biagioni, Hadi Bayat, E. Hui Clarissa Lee, Mohammad Hashemabadi, Hojjat Samareh Fekri, Ali Zarrabi, Reza Mohammadinejad, and Alan Prem Kumar. 2021. "Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives" International Journal of Molecular Sciences 22, no. 21: 11321. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111321

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