Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. (22nd December 2017)

• Record Type: Journal Article
• Authors: LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F.; Jacob, Yannick
• Is Part Of: Plant journal. Volume 93:Number 2(2018)
• Page Start: 377
• Page End: 386
• Publication Date: 2017-12-22
• Subjects: Arabidopsis thaliana -- Citrus -- CRISPR/Cas9 -- heat stress -- off‐target mutations -- SpCas9 -- targeted mutagenesis -- temperature -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.13782
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: 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. Significance Statement: Our study shows that growth temperature can be used to modulate the efficiency of targeted mutagenesis by CRISPR/Cas9 in plants. Our results should help all plant biologists performing CRISPR/Cas9 experiments.