Genetic transformation of plants often results in multiple copies of the introduced DNA at a single locus. To ensure that only a single copy of a foreign gene resides in the plant genome, we used a strategy based on site-specific recombination. The transformation vector consists of a transgene flanked by recombination sites in an inverted orientation. Regardless of the number of copies integrated between the outermost transgenes, recombination between the outermost sites resolves the integrated molecules into a single copy. An example of this strategy has been demonstrated with wheat transformation, where four of four multiple-copy loci were resolved successfully into single-copy transgenes.

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 sgRNA, 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.