Harnessing noncanonical crRNA for highly efficient genome editing
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
DOI:
10.1038/s41467-024-48012-x
Publication Date:
2024-05-07T16:01:52Z
AUTHORS (6)
ABSTRACT
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.
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