High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Frontiers in genome editing Pub Date : 2023-01-01 DOI:10.3389/fgeed.2023.1196763
Ellen Slaman, Michiel Lammers, Gerco C Angenent, Ruud A de Maagd
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引用次数: 1

Abstract

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.

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高通量sgRNA检测揭示了番茄细胞中Cas9特异性和DNA修复的规律。
CRISPR/Cas9技术具有显著提高植物育种的潜力。为了确定SpCas9在番茄中的特异性和致突变谱,我们设计了89 g(uide) rna靶向番茄MYB转录因子家族的基因,这些基因具有不同的预测特异性。将编码sgRNAs和Cas9的质粒导入番茄原生质体,利用扩增子测序技术筛选出突变发生的靶位点和224个预测脱靶位点。在该系统中,用于预测sgrna疗效的算法几乎没有预测能力。突变分析表明,单碱基插入具有可预测的同一性。89个sgrna中有13个发现脱靶突变,并且只发生在一个或两个错配的位置(分别在14个和3个位点)。我们发现pam -近端不匹配不能排除低频脱靶突变。在所有138个有三到四个错配的位置上都没有发现脱靶突变。我们比较了编码sgRNAs和Cas9的质粒与核糖核蛋白(RNP)转染诱导的脱靶突变频率。RNPs的使用导致17个位点中6个位点的相对脱靶频率显著降低,9个位点无显著差异,2个位点增加。此外,我们表明,与sgRNA相关的插入或缺失的脱靶序列可能发生突变,在sgRNA设计时应考虑到这一点。总之,我们的数据通过深入了解cas9诱导的双链断裂修复结果和脱靶突变的发生,帮助sgRNA设计。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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7.00
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审稿时长
13 weeks
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