The heat is on: a simple method to increase genome editing efficiency in plants

Abstract

Background Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. Results Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. Conclusions Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

A Simple and Low‐Tech Heat‐Shock Method to Increase Genome Editing Efficiency in Plants

“…In our experience, 3×HS increases the frequency and/or severity of pds3 phenotypes for different nucleases, plant species, and vector architectures. For example, we observed an increase in the number and/or severity of pds3 phenotypes after 3×HS treatment for ZmUBI-Cas9 (6/9 independent lines), RPS5A-Cas9 (14/14 independent lines), 35S-Cas9 (6/12 independent lines), and PcUBI-Cas9 (7/9 independent lines; (Blomme et al, 2022)).…”

“…For six other T1 lines, heat stress induced an increase in indels: AT2G22460 (+27%), GL1-2 (+57%), IM-1 (+44%), TIR1 (+66%), VAR1-1 (+27%), and VAR1-2 (+55%). We also quantified a 22%-27% increase in C>T base editing efficiency after 3×HS using EDITR (Blomme et al, 2022).…”

“…For example, the white spots or mosaic sectors on leaves of phytoene desaturase3 (pds3; AT4G14210) plants are difficult to observe and easily obscured by faster-growing plants. These issues prompted us to develop an alternative, low-tech methodology that allows for a straightforward induction of heat stress in plants grown on sterile media (Blomme et al, 2022).…”

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora

“…It is also worth mentioning that the temperature difference could have increased the editing efficiency since Cas9 nuclease activity is affected by temperature. Heat treatments 23 , 24 and temporary increases in temperature during incubation 25 are consistently effective at increasing the number of somatic mutations using different CRISPR systems. This gain in efficiency is related to the higher temperatures being closer to the optimal growth temperature of S. pyogenes (40 °C) 24 .…”

And … cut! Identifying chromatin features affecting CRISPR–Cas9 activity in plants

“…CRISPR–Cas9 technology has the major advantage of generating mutations at a very specific, well-targeted site, compared to the classical approaches using random mutagenesis. Although the CRISPR technology has been successfully used for editing both crop genomes and model plants ( Dima et al, 2022 ), editing efficiency can vary greatly ( Kurokawa et al, 2021 ; Blomme et al, 2022 ). The underlying reasons of such variations in efficacy are not fully understood.…”