Genetic variation may confound analysis of CRISPR-Cas9 off-target mutations.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang G
PROVIDER: S-EPMC5902475 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
Publications
Genetic variation may confound analysis of CRISPR-Cas9 off-target mutations.
Wang Guanqun G Du Meijie M Wang Jianbin J Zhu Ting F TF
Cell discovery 20180417
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Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.
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Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.
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Project description:The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9-edited plants. To identify on- and off-target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9-edited cotton plants targeted to three genes, and three negative (Ne) control and three wild-type (WT) plants. In total, 4188-6404 unique single-nucleotide polymorphisms (SNPs) and 312-745 insertions/deletions (indels) were detected in 14 Cas9-edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer-adjacent motif (PAM), we demonstrated that the most variations following Cas9-edited are due either to somaclonal variation or/and pre-existing/inherent variation from maternal plants, but not off-target effects. Of a total of 4413 potential off-target sites (allowing ≤5 mismatches within the 20-bp sgRNA and 3-bp PAM sequences), the WGS data revealed that only four are bona fide off-target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off-target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off-target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.
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