Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish
The knockout efficiency of the CRISPR/Cas9 system largely relies on the frameshift mutation caused by random insertions or deletions at target site of coding DNA sequence (CDS). Splice site (SS) mutations result in mRNA mis-splicing, making gRNAs targeting SS more likely to produce null mutations an...
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Elsevier
2025-09-01
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| Series: | Aquaculture Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352513425003813 |
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| author | Zhen Xu Ma Zhuo Leihui Wang Shuo Yan Shiyi Zhang Ying Chen Guang Xu Chenxu Wang Wenjing Tao Deshou Wang |
| author_facet | Zhen Xu Ma Zhuo Leihui Wang Shuo Yan Shiyi Zhang Ying Chen Guang Xu Chenxu Wang Wenjing Tao Deshou Wang |
| author_sort | Zhen Xu |
| collection | DOAJ |
| description | The knockout efficiency of the CRISPR/Cas9 system largely relies on the frameshift mutation caused by random insertions or deletions at target site of coding DNA sequence (CDS). Splice site (SS) mutations result in mRNA mis-splicing, making gRNAs targeting SS more likely to produce null mutations and thereby enhancing CRISPR efficiency. In this study, gRNAs were designed to target both the CDS and SS of tyrb, hps4, hps5, chrd, eda, fgfr1a, fgf8a and bmp2b in tilapia. Remarkably higher null mutation rate was observed in F0 mutants injected with gRNAs targeting SS compared to those targeting CDS. By transcriptome analysis, E2 skipping, I5 retention and I7 retention were observed in tyrb I2 5’SS, hps4 I5 3’SS and hps5 I7 5’SS mutation, respectively. Furthermore, the phenotype rates were counted for F0 of tyrb (53.8 %/6.9 %), hps5 (78.4 %/0 %) and chrd (49.38 %/16.18 %), and remarkably higher phenotype rates were observed in fish injected gRNAs targeting SS than those targeting CDS. For tyrb and hps5, around 30 % of F0 fish displayed the albino phenotypes as observed in homozygous mutants when targeting SS, whereas those targeting CDS exhibited only a partial reduction of pigmented melanophores on their body surfaces. Mutations in any of the four bases (“NNGT” or “AGNN”) at the exon-intron boundary resulted in mRNA mis-splicing. The CRISPR/cas9 mediated SS mutation was most efficient when the target was selected to produce a DSB within 4 bp flanking the exon-intron boundary. These results demonstrated that gRNAs targeting SS provided an alternative and effective option for gene mutation and new germplasm creation in fish. |
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| institution | Matheson Library |
| issn | 2352-5134 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
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| series | Aquaculture Reports |
| spelling | doaj-art-1a2d0ee2d7cc420f90f4131b5b7c1da52025-07-26T05:23:33ZengElsevierAquaculture Reports2352-51342025-09-0143102995Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fishZhen Xu0Ma Zhuo1Leihui Wang2Shuo Yan3Shiyi Zhang4Ying Chen5Guang Xu6Chenxu Wang7Wenjing Tao8Deshou Wang9Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaIntegrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaCorresponding authors.; Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaCorresponding authors.; Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, ChinaThe knockout efficiency of the CRISPR/Cas9 system largely relies on the frameshift mutation caused by random insertions or deletions at target site of coding DNA sequence (CDS). Splice site (SS) mutations result in mRNA mis-splicing, making gRNAs targeting SS more likely to produce null mutations and thereby enhancing CRISPR efficiency. In this study, gRNAs were designed to target both the CDS and SS of tyrb, hps4, hps5, chrd, eda, fgfr1a, fgf8a and bmp2b in tilapia. Remarkably higher null mutation rate was observed in F0 mutants injected with gRNAs targeting SS compared to those targeting CDS. By transcriptome analysis, E2 skipping, I5 retention and I7 retention were observed in tyrb I2 5’SS, hps4 I5 3’SS and hps5 I7 5’SS mutation, respectively. Furthermore, the phenotype rates were counted for F0 of tyrb (53.8 %/6.9 %), hps5 (78.4 %/0 %) and chrd (49.38 %/16.18 %), and remarkably higher phenotype rates were observed in fish injected gRNAs targeting SS than those targeting CDS. For tyrb and hps5, around 30 % of F0 fish displayed the albino phenotypes as observed in homozygous mutants when targeting SS, whereas those targeting CDS exhibited only a partial reduction of pigmented melanophores on their body surfaces. Mutations in any of the four bases (“NNGT” or “AGNN”) at the exon-intron boundary resulted in mRNA mis-splicing. The CRISPR/cas9 mediated SS mutation was most efficient when the target was selected to produce a DSB within 4 bp flanking the exon-intron boundary. These results demonstrated that gRNAs targeting SS provided an alternative and effective option for gene mutation and new germplasm creation in fish.http://www.sciencedirect.com/science/article/pii/S2352513425003813CRISPR/Cas9gRNA targeting splice siteExon skippingIntron retentionNull mutation and phenotype rate |
| spellingShingle | Zhen Xu Ma Zhuo Leihui Wang Shuo Yan Shiyi Zhang Ying Chen Guang Xu Chenxu Wang Wenjing Tao Deshou Wang Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish Aquaculture Reports CRISPR/Cas9 gRNA targeting splice site Exon skipping Intron retention Null mutation and phenotype rate |
| title | Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish |
| title_full | Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish |
| title_fullStr | Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish |
| title_full_unstemmed | Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish |
| title_short | Splice-site mutation enhances the efficiency of CRISPR-mediated gene knockout and new germplasm creation in fish |
| title_sort | splice site mutation enhances the efficiency of crispr mediated gene knockout and new germplasm creation in fish |
| topic | CRISPR/Cas9 gRNA targeting splice site Exon skipping Intron retention Null mutation and phenotype rate |
| url | http://www.sciencedirect.com/science/article/pii/S2352513425003813 |
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