Harnessing noncanonical crRNA for highly efficient genome editing

• DOI: 10.1038/s41467-024-48012-x
• Publication Date: 2024-05-07T16:01:52Z
• Authors: Guanhua Xun; Zhixin Zhu; Nilmani Singh; Jingxia Lu; Piyush K. Jain; Huimin Zhao
• Keywords: Gene Editing; 0303 health sciences; Endodeoxyribonucleases; Science; Q; CRISPR-Associated Proteins; RNA, Guide, CRISPR-Cas Systems; Article; 03 medical and health sciences; HEK293 Cells; Bacterial Proteins; Humans; RNA; CRISPR-Cas Systems

AbstractThe CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome 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.