Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Abstract

Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock inducible CRISPR/Cas9 system for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using a soybean heatshock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS) inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted by transforming rice with HS-CRISPR/Cas9 vectors, and the presence of targeted mutations was determined before and after the HS treatment. We found only a low rate of targeted mutagenesis before HS, but an increased rate of mutagenesis after HS treatment among the transgenic lines. Specifically, only ~11% of transformants showed characteristic insertions-deletions at the ambient room temperature, but a higher percentage (~45%) of callus lines developed mutations after a few days of HS treatment. Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after several weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutant lines that independently segregated from Cas9. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing. Significance Statement:A method for the temporal control on gene editing based on the use of heat-shock induced expression of CRISPR/Cas9 is described, which was efficient in producing heritable mutations in the rice genome. We assume this method will be useful for targeting essential genes and improving the precision of CRISPR/Cas9.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

“…Another interesting result of this study is the low targeting efficiency of HS-inducible Cas9 compared to constitutive Cas9 (Table 1 ). This finding was also appreciated by Nandy et al 36 , who compared the relative expressions of HS inducible- versus constitutive-Cas9 and found that the latter was 800 times more expressed than the former. The low expression level of inducible Cas9 can be partially balanced by its higher enzymatic activity at the high temperature used during the induction step, close to 40°C 37 .…”

“…A basal expression of Cas9 (not yet induced by the treatment) was likely responsible for the DNA cleavage at CTS (between the third and the fourth nucleotide from the PAM site) with the outcome of some small deletions. Nandy and colleagues 36 , who applied heat‐shock-inducible CRISPR/Cas9 system in rice, detected a rate of basal targeted mutagenesis (before HS) around 16% (two out of 12 lines showed editing in one target site; 1 out of 6 lines in a second target site). This was quite unexpected because when used to drive expression of recombinase enzymes, the soybean heat-shock promoter proved to be tightly regulated by heat treatment 15 , 23 .…”

Abiotic Stress Responses in Plants: Current Knowledge and Future Prospects

“…However, one major limitation of genome editing is the off-target mutations that are caused by Cas9 in transgenic plants. This limitation has been overcome to a considerable extent by the advent of stress-inducible CRISPR/Cas9 technique which reduces the rate of off-target mutations to negligible levels [112]. Thus, we can consider stressinducible CRISPR/Cas as a promising tool for precise and efficient genome editing in crop plants for numerous traits, including abiotic stress tolerance.…”

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

“…Adaptations to transformation vectors are typically used to increase genome editing efficiency, such as using different constitutive or germline‐specific promoters to control Cas9 expression or optimization of other regulatory sequences such as terminators, introns, or Pol III promoters that drive gRNA expression (Feng et al., 2018; Mao et al., 2016; Ordon et al., 2020; Tsutsui & Higashiyama, 2017; Wang et al., 2015; Yan et al., 2015). In addition to optimizing transformation vectors, mutagenesis efficiency can be increased by applying small molecules in mammalian cells (Yu et al., 2015) or applying heat in a variety of organisms (Greiner et al., 2017; LeBlanc et al., 2018; Li et al., 2021; Milner, Craze, Hope, & Wallington, 2020; Moreno‐Mateos et al., 2017; Nandy, Pathak, Zhao, & Srivastava, 2019; Vu et al., 2020; Xiang, Zhang, An, Cheng, & Wang, 2017). Although heat treatment can induce genome editing efficiency in plants, most published experimental setups make use of dedicated growth chambers to apply the heat treatment (An et al., 2020; Kurokawa et al., 2021; LeBlanc et al., 2018; Li et al., 2021; Milner et al., 2020; Nandy et al., 2019; Vu et al., 2020).…”

“…In addition to optimizing transformation vectors, mutagenesis efficiency can be increased by applying small molecules in mammalian cells (Yu et al., 2015) or applying heat in a variety of organisms (Greiner et al., 2017; LeBlanc et al., 2018; Li et al., 2021; Milner, Craze, Hope, & Wallington, 2020; Moreno‐Mateos et al., 2017; Nandy, Pathak, Zhao, & Srivastava, 2019; Vu et al., 2020; Xiang, Zhang, An, Cheng, & Wang, 2017). Although heat treatment can induce genome editing efficiency in plants, most published experimental setups make use of dedicated growth chambers to apply the heat treatment (An et al., 2020; Kurokawa et al., 2021; LeBlanc et al., 2018; Li et al., 2021; Milner et al., 2020; Nandy et al., 2019; Vu et al., 2020). Such chambers might not be regularly available to a wide range of laboratories, and are notorious for temperature fluctuations.…”