Department of Molecular, Cellular and Developmental Biology, Faculty of Arts and Sciences, Yale University, 219 Prospect Street, New Haven, CT, 06511, USA.
Plant J. 2018 Jan;93(2):377-386. doi: 10.1111/tpj.13782. Epub 2017 Dec 22.
The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.
CRISPR/Cas9 系统极大地提高了我们在真核基因组中工程靶向突变的能力。虽然 CRISPR/Cas9 似乎普遍适用，但靶向诱变的效率和脱靶突变的不利产生在不同的生物体之间差异很大。在这项研究中，我们报告称，在 37°C 下经受热应激的拟南芥植物比在标准温度（22°C）下连续生长的植物表现出更高频率的 CRISPR 诱导突变。使用依赖于绿色荧光蛋白（GFP）报告基因的定量测定，我们发现 CRISPR/Cas9 在拟南芥体细胞组织中的靶向突变增加了约 5 倍，在生殖细胞中增加了高达 100 倍。这种温度对突变率的影响不仅限于拟南芥，因为我们在暴露于 37°C 热应激的柑橘植物中观察到 CRISPR/Cas9 的靶向突变也有类似的增加。体外实验表明，来自酿脓链球菌（SpCas9）的 Cas9 在 37°C 下比在 22°C 下更有效地产生双链 DNA 断裂，因此表明温度对 CRISPR/Cas9 的体内效应的潜在贡献机制。这项研究揭示了温度在调节真核生物中 SpCas9 活性方面的重要性，并提供了一种使用 CRISPR/Cas9 增加植物中靶突变的简单方法。
