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 heat-shock 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.


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