Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency.
basic_science · Level V
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- Record sourced from PubMed, PMID 28840077.
- Also identified by DOI 10.1038/s41551-017-0066 and PMC identifier 5562407.
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Abstract
Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered <i>Acidaminococcus sp.</i> Cpf1 (AsCpf1) CRISPR RNAs (crRNAs) and 5 types of AsCpf1 mRNAs, and show that the top-performing modified crRNA (cr3'5F, containing five 2'-fluoro ribose at the 3' termini) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3'5F and ψ-modified AsCpf1 or <i>Lachnospiraceae bacterium</i> Cpf1 (LbCpf1) mRNAs augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologs enabled genome editing in the presence of AsCpf1. Engineered CRISPR-Cpf1 systems should facilitate a broad range of genome editing applications.