Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep
Abstract Background The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2018-05-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-018-4712-z |
| _version_ | 1818267096766742528 |
|---|---|
| author | Xiaolong Wang Jing Liu Yiyuan Niu Yan Li Shiwei Zhou Chao Li Baohua Ma Qifang Kou Bjoern Petersen Tad Sonstegard Xingxu Huang Yu Jiang Yulin Chen |
| author_facet | Xiaolong Wang Jing Liu Yiyuan Niu Yan Li Shiwei Zhou Chao Li Baohua Ma Qifang Kou Bjoern Petersen Tad Sonstegard Xingxu Huang Yu Jiang Yulin Chen |
| author_sort | Xiaolong Wang |
| collection | DOAJ |
| description | Abstract Background The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. Results To address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence. Conclusions We provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9. |
| first_indexed | 2024-12-12T20:17:10Z |
| format | Article |
| id | doaj.art-8acf8044cee840d4814a0d901652a6df |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-12T20:17:10Z |
| publishDate | 2018-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-8acf8044cee840d4814a0d901652a6df2022-12-22T00:13:21ZengBMCBMC Genomics1471-21642018-05-011911810.1186/s12864-018-4712-zLow incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheepXiaolong Wang0Jing Liu1Yiyuan Niu2Yan Li3Shiwei Zhou4Chao Li5Baohua Ma6Qifang Kou7Bjoern Petersen8Tad Sonstegard9Xingxu Huang10Yu Jiang11Yulin Chen12College of Animal Science and TechnologyCollege of Animal Science and TechnologyCollege of Animal Science and TechnologyCollege of Animal Science and TechnologyCollege of Animal Science and TechnologyCollege of Animal Science and TechnologyCollege of Veterinary Medicine, Northwest A&F UniversityNingxia Tianyuan Tan Sheep FarmInstitute of Farm Animal Genetics, Friedrich-Loeffler-InstitutRecombinetics, St. PaulSchool of Life Science and Technology, ShanghaiTech UniversityCollege of Animal Science and TechnologyCollege of Animal Science and TechnologyAbstract Background The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. Results To address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence. Conclusions We provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.http://link.springer.com/article/10.1186/s12864-018-4712-zGenome editingCRISPR/Cas9Whole genome sequencingOff-target; mutation rateSheep |
| spellingShingle | Xiaolong Wang Jing Liu Yiyuan Niu Yan Li Shiwei Zhou Chao Li Baohua Ma Qifang Kou Bjoern Petersen Tad Sonstegard Xingxu Huang Yu Jiang Yulin Chen Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep BMC Genomics Genome editing CRISPR/Cas9 Whole genome sequencing Off-target; mutation rate Sheep |
| title | Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep |
| title_full | Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep |
| title_fullStr | Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep |
| title_full_unstemmed | Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep |
| title_short | Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep |
| title_sort | low incidence of snvs and indels in trio genomes of cas9 mediated multiplex edited sheep |
| topic | Genome editing CRISPR/Cas9 Whole genome sequencing Off-target; mutation rate Sheep |
| url | http://link.springer.com/article/10.1186/s12864-018-4712-z |
| work_keys_str_mv | AT xiaolongwang lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT jingliu lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT yiyuanniu lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT yanli lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT shiweizhou lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT chaoli lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT baohuama lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT qifangkou lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT bjoernpetersen lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT tadsonstegard lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT xingxuhuang lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT yujiang lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep AT yulinchen lowincidenceofsnvsandindelsintriogenomesofcas9mediatedmultiplexeditedsheep |