Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a.

The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.