The CRISPR / C as system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in A rabidopsis resulting in heritable progeny

Summary The CRISPR / C as nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double‐strand breaks ( DSB s) at pre‐selected genomic sites that are repaired by non‐homologous end joining ( NHEJ ) in an error‐prone way. In plants, it could be demonstrated that the C as9 nuclease is able to induce heritable mutations in A rabidopsis thaliana and rice. Gene targeting ( GT ) by homologous recombination ( HR ) can also be induced by DSB s. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR / C as‐mediated DSB induction. We were able to integrate a resistance cassette into the ADH 1 locus of A . thaliana via HR . Heritable events were identified using a PCR ‐based genotyping approach, characterised by S outhern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR / C as nucleases. Off‐target effects might be avoided using two adjacent sg RNA target sequences to guide the C as9 nickase to each of the two DNA strands, resulting in the formation of a DSB . By amplicon deep sequencing, we demonstrate that this C as9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A . thaliana .