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.

Mapping the adaptor protein complex interaction network in Arabidopsis identifies P34 as a common stability regulator

“…To this end, we crossed the p34 iCRISPR -1 mutant with pAP2S:AP2S-GFP/ap2s , pAP3B:AP3B-GFP/pat2-1 , and pAP4M:AP4M-GFP/ap4m and used the p35S-GFP /Col-0 plants as control. F2 seeds carrying the CRISRP/Cas9 construct were selected by seed fluorescence 49 and used for further experiments. Immunoblot analysis indicated that the levels of AP2S-GFP and AP4M-GFP, but not of AP3B-GFP and GFP, were reduced after β-estradiol induction (Fig.…”

“…1 and Supplemental Data 1) and all three AP-5 subunits were identified together with the thus far uncharacterized proteins in plants ZINC FINGER FYVE DOMAIN-CONTAINING PROTEIN (ZFYVE) (AT2G25730) and SPATACSIN CARBOXY-TERMINUS PROTEIN (AT4G39420). Further sequence alignments revealed the homology of the SPATACSIN CARBOXY-TERMINUS PROTEIN and the ZFYVE protein to the mammalian SPG11 (with 60% similarity and 22.83% identity) and SPG15 (with 49% similarity and 22.57 identity) proteins, respectively, the two known accessory proteins of the AP-5 complex in mammalian cells 2, 49 . Consistent with the AP-3 and AP-4 interactome analyses, AP5M co-purified with four AP-4 subunits (Fig.…”

“…The two gRNAs (Supplementary Table 1) were cloned into the entry vectors pGG-C-AtU6-26-BbsI-ccdB-BbsI-D and pGG-D-AtU6-26-BbsI-ccdB-BbsI-E according to the previously described protocol 71 . The CRISPR/Cas9 expression construct was generated by assembling the entry clones with pGG-A-linkerIII-C , pGG-E-linker-G 92 , into the backbone vector pFASTRK-Atcas9-AG 49 with the Golden Gate reaction. For the inducible CRISPR construct, the entry clones of the gRNAs and pGG-A-linkerIII-C , pGG-E-linker-G were first cloned into the pEN-R2-AG-L3 74 with the Golden Gate reaction.…”

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

“…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).…”

“…In this methodology article, we present a detailed description of our simple and straightforward setup to impose heat stress on Arabidopsis and tobacco using commonly available laboratory equipment (Basic Protocol 1; Blomme et al., 2022). Our simplified three heat shock (3×HS) protocol allows researchers to increase genome editing efficiencies in the T1 generation by 15%‐66%, depending on the target site, for many CRISPR applications.…”