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

Summary 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 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat‐shock protein gene promoter and the rice U3 promoter to express Cas9 and sg RNA , respectively, we developed the heat‐shock ( HS )‐inducible CRISPR /Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50–63%). 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 a few weeks of HS treatments. Most importantly, the HS ‐induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off‐target mutations were either undetectable or found at a lower rate in HS ‐ CRISPR /Cas9 lines as compared to the constitutive‐overexpression CRISPR /Cas9 lines. 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.