A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice

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dc.contributor.authorTang, Xu
dc.contributor.authorLiu, Guanqing
dc.contributor.authorZhou, Jianping
dc.contributor.authorRen, Qiurong
dc.contributor.authorYou, Qi
dc.contributor.authorTian, Li
dc.contributor.authorXin, Xuhui
dc.contributor.authorZhong, Zhaohui
dc.contributor.authorLiu, Binglin
dc.contributor.authorZheng, Xuelian
dc.contributor.authorZhang, Dengwei
dc.contributor.authorMalzahn, Aimee
dc.contributor.authorGong, Zhiyun
dc.contributor.authorQi, Yiping
dc.contributor.authorZhang, Tao
dc.contributor.authorZhang, Yong
dc.date.accessioned2021-06-28T19:45:03Z
dc.date.available2021-06-28T19:45:03Z
dc.date.issued2018-07-04
dc.description.abstractTargeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date. We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.en_US
dc.description.urihttps://doi.org/10.1186/s13059-018-1458-5
dc.identifierhttps://doi.org/10.13016/k6ws-gnac
dc.identifier.citationTang, X., Liu, G., Zhou, J. et al. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol 19, 84 (2018).en_US
dc.identifier.urihttp://hdl.handle.net/1903/27178
dc.language.isoen_USen_US
dc.publisherSpringer Natureen_US
dc.relation.isAvailableAtCollege of Agriculture & Natural Resourcesen_us
dc.relation.isAvailableAtPlant Science & Landscape Architectureen_us
dc.relation.isAvailableAtDigital Repository at the University of Marylanden_us
dc.relation.isAvailableAtUniversity of Maryland (College Park, MD)en_us
dc.subjectGenome Editingen_US
dc.subjectSingle Nucleotide Variants (SNVs)en_US
dc.subjectCas9 sgRNAen_US
dc.subjectIndelsen_US
dc.subjectRNA-guided Nucleases (RGN)en_US
dc.titleA large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in riceen_US
dc.typeArticleen_US

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