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Genetic Analysis Based on CRISPR/Cas9 Technology
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Genetic Analysis Based on CRISPR/Cas9 Technology

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

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 14421

Special Issue Editors

Prof. Dr. Kwon-Kyoo Kang

E-Mail Website
Guest Editor
Division of Horticultural Biotechnology, Hankyung National University, Anseong 17579, Republic of Korea
Interests: functional analysis of genes via CRISPR/Cas9; functional genomics; MABc (marker-assisted backcross); transcriptomics; plant biotechnology; molecular breeding in plants
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Yong-Gu Cho

E-Mail Website
Guest Editor
Department of Crop Science, College of Agriculture, Life and Environment Sciences, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Chongju 28644, Republic of Korea
Interests: marker-assisted breeding (MAS); plant breeding by CRISPR/Cas9; functional genomics; GWAS; biotic and abiotic tolerance; transcriptomics; functional analysis of genes; plant biotechnology; molecular breeding in rice
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Recently, many papers have been reported on the results in which gene editing technology was applied using functional genes of plants and animals. These reports suggest that gene editing technology has excellent genome editing tools and is applicable to most organisms. It is interesting on how mutations are obtained through gene editing such as KO, KI, NHEJ and HDR, and how is affected the expression of many other genes in the genome as well as genetic analysis.  We will be accepting on papers how to use the genetic resources to solve genetic problems and how to apply them to new breeding programs through gene editing technology. This Special Issue of the International Journal of Molecular Sciences (IJMS) welcomes to have articles on reviews and research results.

Prof. Dr. Kwon-Kyoo Kang
Prof. Dr. Yong-Gu Cho
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Gene targeting (GT)
  • Homology-directed repair (HDR)
  • Homology-directed gene targeting (HGT)
  • CRISPR/Cas
  • Targeted mutagenesis
  • Precision breeding
  • genetic analysis

Published Papers (7 papers)

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Editorial

Jump to: Research

3 pages, 189 KiB  
Editorial
Genetic Analysis Based on CRISPR/Cas9 Technology in Plants
by Kwon-Kyoo Kang and Yong-Gu Cho
Int. J. Mol. Sci. 2023, 24(22), 16398; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242216398 - 16 Nov 2023
Viewed by 652
Abstract
Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...] Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)

Research

Jump to: Editorial

22 pages, 10999 KiB  
Article
Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice
by Me-Sun Kim, Seo-Rin Ko, Yu Jin Jung, Kwon-Kyoo Kang, Yung-Jo Lee and Yong-Gu Cho
Int. J. Mol. Sci. 2023, 24(6), 5338; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24065338 - 10 Mar 2023
Cited by 5 | Viewed by 2029
Abstract
Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for [...] Read more.
Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2 OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein–protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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14 pages, 3508 KiB  
Article
Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System
by Jin Young Kim, Jong Hee Kim, Young Hee Jang, Jihyeon Yu, Sangsu Bae, Me-Sun Kim, Yong-Gu Cho, Yu Jin Jung and Kwon Kyoo Kang
Int. J. Mol. Sci. 2023, 24(1), 109; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24010109 - 21 Dec 2022
Cited by 7 | Viewed by 2312
Abstract
Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene [...] Read more.
Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography–tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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15 pages, 8608 KiB  
Article
CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis)
by Yun-Hee Shin, Hyun-Min Lee and Young-Doo Park
Int. J. Mol. Sci. 2022, 23(23), 15009; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315009 - 30 Nov 2022
Cited by 5 | Viewed by 1728
Abstract
Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting—the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced [...] Read more.
Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting—the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0 AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line ‘CT001.’ Generational progression using bud pollination obtained T-DNA-free E1 AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis). Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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10 pages, 3495 KiB  
Article
Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice
by Jong Hee Kim, Jihyeon Yu, Hee Kyoung Kim, Jin Young Kim, Me-Sun Kim, Yong-Gu Cho, Sangsu Bae, Kwon Kyoo Kang and Yu Jin Jung
Int. J. Mol. Sci. 2022, 23(18), 10383; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810383 - 08 Sep 2022
Cited by 7 | Viewed by 1940
Abstract
Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have [...] Read more.
Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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16 pages, 2256 KiB  
Article
Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9
by Na-Ri Shin, Yun-Hee Shin, Han-Seul Kim and Young-Doo Park
Int. J. Mol. Sci. 2022, 23(9), 5062; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23095062 - 03 May 2022
Cited by 8 | Viewed by 2044
Abstract
Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, [...] Read more.
Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50–70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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13 pages, 1218 KiB  
Article
Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco α-1,3-Fucosyltransferase 1 and β-1,2-Xylosyltransferase 1 Loci
by Hayoung Song, Ju-Young Ahn, Fanzhuang Yan, Yidong Ran, Okjae Koo and Geung-Joo Lee
Int. J. Mol. Sci. 2022, 23(5), 2450; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052450 - 23 Feb 2022
Cited by 4 | Viewed by 2037
Abstract
We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and [...] Read more.
We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37–54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a −4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis. Full article
(This article belongs to the Special Issue Genetic Analysis Based on CRISPR/Cas9 Technology)
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