(236c) Harnessing Noncanonical Crrna for Highly Efficient Genome Editing

The CRISPR-Cas system has been extensively studied for its potential in genome editing and gene regulation across a variety of organisms over the last decade. Despite its promise, the widely used CRISPR-Cas9 nuclease, which is derived from Streptococcus pyogenes, has been found to have limited application due to its tendency to cause off-target editing effects in eukaryotic cells. In contrast, the Cas12 nuclease derived from Acidaminococcus sp has been found to be more specific and accurate, but its use is limited by its low on-target editing efficiency. Many protein engineering and crRNA engineering strategies have been developed to overcome this limitation, but with limited success. Here we report an engineered highly efficient and precise CRISPR-Cas12a system named zCRISPR-Cas12a that incorporates 2’-aminoadenine (base Z) into the crRNA to increase the binding affinity between crRNA and its complementary DNA. This zCRISPR-Cas12a system shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but much lower off-target effects than that of the CRISPR-Cas9 system. In addition, this system can be used for precise gene knock-in, highly efficient multiplex gene editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.