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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 August 2021) | Viewed by 46772

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Prof. Dr. John Parrington

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Guest Editor

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
Interests: cell signalling; pharmacology; gene editing; genomic analysis; small and large animal models

Prof. Dr. Joaquín Gadea

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Guest Editor

Department of Physiology, International Excellence Campus for Higher Education and Research “Campus Mare Nostrum” and Institute for Biomedical Research of Murcia (IMIB-Arrixaca), University of Murcia, 30100 Murcia, Spain
Interests: animal reproduction; fertility; sperm assestment; gene editing
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Dear Colleagues,

Since the birth of civilization, human beings have manipulated other life forms. We have selectively bred plants and animals for thousands of years to maximize agricultural production and obtain pets according to our tastes. The ability to directly engineer the genomes of organisms first became possible in the 1970s, when the gene for human insulin was introduced into bacteria to produce this protein for diabetics. In the past few years, the pace of progress has accelerated enormously. We can now cut and paste genes using ‘molecular scissors’ with astonishing ease, and the new technology of genome editing can be applied to practically any species of plants or animals.

Genome editing is transforming medical research and is already being tested as a direct treatment for human disease. In agriculture, genome editing could be used to engineer species with increased food output and the ability to thrive in challenging climates. However, these powerful new techniques also raise important ethical dilemmas and potential dangers, pressing issues that we need to face, given the speed of scientific developments.

This Special Issue will consider papers on new scientific developments in genome editing and their latest implications in various areas of biotechnology.

Prof. Dr. John Parrington
Guest Editor

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

genome editing
biotechnology
animal models
biomedical research
drug development
gene therapy

Published Papers (12 papers)

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Research

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11 pages, 1743 KiB

Open AccessArticle

Proviral ALV-LTR Sequence Is Essential for Continued Proliferation of the ALV-Transformed B Cell Line

by Swagata Roy, Megha Sravani Bondada, Yaoyao Zhang, Katy Moffat, Venugopal Nair and Yongxiu Yao

Int. J. Mol. Sci. 2022, 23(19), 11263; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911263 - 24 Sep 2022

Cited by 1 | Viewed by 1519

Abstract

Avian leukosis virus (ALV) induces B-cell lymphomas and other malignancies in chickens through insertional activation of oncogenes, and c-myc activation has been commonly identified in ALV-induced tumors. Using ALV-transformed B-lymphoma-derived HP45 cell line, we applied in situ CRISPR-Cas9 editing of integrated proviral long terminal repeat (LTR) to examine the effects on gene expression and cell proliferation. Targeted deletion of LTR resulted in significant reduction in expression of a number of LTR-regulated genes including c-myc. LTR deletion also induced apoptosis of HP45 cells, affecting their proliferation, demonstrating the significance of LTR-mediated regulation of critical genes. Compared to the global effects on expression and functions of multiple genes in LTR-deleted cells, deletion of c-myc had a major effect on the HP45 cells proliferation with the phenotype similar to the LTR deletion, demonstrating the significance of c-myc expression in ALV-induced lymphomagenesis. Overall, our studies have not only shown the potential of targeted editing of the LTR for the global inhibition of retrovirus-induced transformation, but also have provided insights into the roles of LTR-regulated genes in ALV-induced neoplastic transformation. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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15 pages, 5319 KiB

Open AccessArticle

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation

by Ahmed K. Mahdi, Juan F. Medrano and Pablo J. Ross

Int. J. Mol. Sci. 2022, 23(18), 10218; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810218 - 06 Sep 2022

Cited by 2 | Viewed by 1896

Abstract

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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16 pages, 2737 KiB

Open AccessArticle

Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9

by Sergio Navarro-Serna, Celia Piñeiro-Silva, Chiara Luongo, John Parrington, Raquel Romar and Joaquín Gadea

Int. J. Mol. Sci. 2022, 23(4), 2135; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23042135 - 15 Feb 2022

Cited by 6 | Viewed by 2237

Abstract

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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15 pages, 2372 KiB

Open AccessArticle

Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in Drosophila suzukii

by Ying Yan, Yukino Kobayashi, Cong Huang, Bo Liu, Wanqiang Qian, Fanghao Wan and Marc F. Schetelig

Int. J. Mol. Sci. 2021, 22(13), 6724; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22136724 - 23 Jun 2021

Cited by 7 | Viewed by 3173

Abstract

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3’UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G₀ flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G₀ flies produced G₁ mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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15 pages, 2294 KiB

Open AccessArticle

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

by Nina Reuven, Julia Adler, Nadav Myers and Yosef Shaul

Int. J. Mol. Sci. 2021, 22(7), 3741; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073741 - 03 Apr 2021

Cited by 9 | Viewed by 4513

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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12 pages, 2119 KiB

Open AccessArticle

Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

by Sung Wook Shin, Dongwoo Kim and Jae Seong Lee

Int. J. Mol. Sci. 2021, 22(5), 2407; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052407 - 27 Feb 2021

Cited by 7 | Viewed by 3051

Abstract

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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14 pages, 2632 KiB

Open AccessArticle

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

by Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi

Int. J. Mol. Sci. 2021, 22(5), 2249; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052249 - 24 Feb 2021

Cited by 19 | Viewed by 2916

Abstract

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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Review

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17 pages, 1295 KiB

Open AccessReview

Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases

by Juliët Schreurs, Claudia Sacchetto, Robin M. W. Colpaert, Libero Vitiello, Alessandra Rampazzo and Martina Calore

Int. J. Mol. Sci. 2021, 22(20), 10985; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222010985 - 12 Oct 2021

Cited by 7 | Viewed by 5573

Abstract

In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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30 pages, 2769 KiB

Open AccessReview

Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies

by Nicolás González Castro, Jan Bjelic, Gunya Malhotra, Cong Huang and Salman Hasan Alsaffar

Int. J. Mol. Sci. 2021, 22(19), 10355; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910355 - 26 Sep 2021

Cited by 25 | Viewed by 7349

Abstract

Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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17 pages, 5041 KiB

Open AccessReview

Points of View on the Tools for Genome/Gene Editing

by Chin-Kai Chuang and Wei-Ming Lin

Int. J. Mol. Sci. 2021, 22(18), 9872; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189872 - 13 Sep 2021

Cited by 9 | Viewed by 2883

Abstract

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H₈₄₀A) and RuvC nuclease activity null mutant (D₁₀A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D₁₀A), Cas9(H₈₄₀A), and Cas9(D₁₀A, H₈₄₀A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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10 pages, 267 KiB

Open AccessReview

Genotype to Phenotype: CRISPR Gene Editing Reveals Genetic Compensation as a Mechanism for Phenotypic Disjunction of Morphants and Mutants

by Cristy M. Salanga and Matthew C. Salanga

Int. J. Mol. Sci. 2021, 22(7), 3472; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073472 - 27 Mar 2021

Cited by 17 | Viewed by 3822

Abstract

Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

26 pages, 2076 KiB

Open AccessReview

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

by Karim Shalaby, Mustapha Aouida and Omar El-Agnaf

Int. J. Mol. Sci. 2020, 21(19), 7353; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197353 - 05 Oct 2020

Cited by 22 | Viewed by 6195

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes. Full article

(This article belongs to the Special Issue Advances in Genome Editing)

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