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5' extended gRNA increases the cleavage activity of SuperFi-Cas9

ABSTRACT: High-fidelity Cas enzyme is pivotal to the safety of CRISPR technology in translational research and clinics. However, increasing editing fidelity often comes at the cost of a significant decrease in nuclease editing efficiency, e.g., the rationale designed SuperFi-Cas9 based on the structural basis for mismatch surveillance of wildtype Cas9. By conducting high-throughput profiling on the editing efficiency of 91,603 gRNAs, we found that SuperFi-Cas9/gRNA showed a strong nucleotide preference at the 21st position of the PAM-distal region of the target. A deep-learning model trained from the profiling data further provided a mutation map, which implied that a gRNA-target duplex with twenty-one matched nucleotides positively contributed to the editing efficiency of SuperFi-Cas9/gRNA. By further measuring the cleavage activities at twenty endogenous genomic locations, we demonstrated that a gRNA with extended 5' nucleotides significantly increased the editing activity of SuperFi-Cas9 while remaining its high fidelity, which makes SuperFi-Cas9 a valuable nuclease of the CRISPR toolbox. Together, by deep-learning modeling on high throughput profiling data, we reported that gRNAs with extended 5' nucleotides can rescue the impaired cleavage activities of SuperFi-Cas9.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279806 | GEO | 2025/12/15

REPOSITORIES: GEO

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5' extended gRNA increases the cleavage activity of SuperFi-Cas9

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