Hi-Fi SaCas9 variant
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB51773 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR9435480_1.fastq.gz | Fastqsanger.gz | |||
| ERR9435480_2.fastq.gz | Fastqsanger.gz | |||
| ERR9435461_1.fastq.gz | Fastqsanger.gz | |||
| ERR9435461_2.fastq.gz | Fastqsanger.gz | |||
| ERR9435593_1.fastq.gz | Fastqsanger.gz |
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Project description:C-to-T base editing mediated by CRISPR/Cas9 base editors (BEs) needs a G/C-rich PAM and the editing fidelity is compromised by unwanted indels and non-C-to-T substitutions. We developed CRISPR/Cpf1-based BEs to recognize a T-rich PAM and induce efficient C-to-T editing with few indels and/or non-C-to-T substitutions. The requirement of editing fidelity in therapeutic-related trials necessitates the development of CRISPR/Cpf1-based BEs, which also facilitates base editing in A/T-rich regions.
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Project description: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.
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