Abstract
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.
Highlights
Sequence-specific nucleases (SSNs) have been widely used for genome editing, and widely used genome editing tools include ZFNs[1], TALENs[2] and, more recently, the CRISPR-associated protein 9 (Cas9) system[3]
We designed four CRISPR/ Cas[9] single guide RNA (sgRNA) based on the sequence of VvbZIP36, a gene that has been shown to play a role in drought stress responses[47] and obtained one mutant grapevine plant
A 52-bp deletion was detected in W52_38 and W52_52 between sgRNA3 and sgRNA4, consistent with the previous results[12]. These results indicated that different sgRNAs can induce different types of mutations and that the most common types of mutations are short insertions and deletions, indicating that the CRISPR/ Cas[9] system can be used for precise genome editing in the grapevine
Summary
Sequence-specific nucleases (SSNs) have been widely used for genome editing, and widely used genome editing tools include ZFNs (zinc finger nucleases)[1], TALENs (transcription activator-like effector nucleases)[2] and, more recently, the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system[3]. The CRISPR/Cas[9] system uses an RNA-protein complex consisting of two essential components: a Cas[9] effector protein and a single guide RNA (sgRNA) containing a targeting sequence of ~20. Cells can only ensure normal activity by repairing DSBs by either nonhomologous end-joining (NHEJ) or homologydirected repair (HDR)[3]. This DSB repair process often leads to on-target and off-target mutations[13]. The latter occurs due to the ability of the sgRNA to recognize genomic sites with a few nucleotide mismatches. The binding and cutting efficiencies are lower when the sgRNA recognizes DNA with mismatches[17]
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