RNA-guided DNA endonuclease from Acidaminococcus sp. BV3L6, formerly named AsCpf1, that cleaves double-stranded DNA at sites specified by a single crRNA and requires a T-rich PAM immediately 5' of the target. It needs no tracrRNA and leaves staggered ends with 5' overhangs. One of two Cpf1-family nucleases, with LbCas12a, that edit human cells robustly.
Length: 4107 bp (1369 aa)
PAM: TTTV
Cut type: 5_overhang
Effector domain: RuvC
Origin: Acidaminococcus sp. BV3L6 type V CRISPR-Cas system
Characteristics
Cleaves both DNA strands with a single RuvC-like nuclease domain, producing a staggered double-strand break with a 4-5 nucleotide 5' overhang distal to the PAM. Processes its own CRISPR array into mature crRNAs without a tracrRNA. Of 16 Cpf1-family proteins screened, only AsCpf1 and LbCpf1 produced robust indels in HEK293FT cells, at levels comparable to Cas9. Mutating the RuvC catalytic residues abolishes cleavage of both strands rather than producing a nickase. Digenome-seq maps 12 cleavage sites per crRNA for AsCas12a, against over 90 for Cas9.
Applications: Genome editing in HEK293FT cells with indel levels comparable to SpCas9 at the tested targets, and multiplexed editing from a single crRNA array. Serves as the parent of the PAM-broadened variant enAsCas12a, which extends targeting beyond the canonical TTTV PAM.
Limitations: The canonical TTTV PAM occurs at a theoretical frequency of 3/256 in DNA against 16/256 for the NGG PAM of SpCas9, leaving far fewer targetable sites; enAsCas12a widens this range. Activity depends on temperature, and AsCas12a works poorly in ectothermic organisms such as zebrafish and Xenopus. Target binding triggers indiscriminate trans cleavage of single-stranded DNA.
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Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.
Cell - Zetsche 2015 Cas12a
Moreno-Mateos et al. (2017).
CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.
Nat Commun - Moreno-Mateos 2017 Cpf1 Temperature
Zetsche et al. (2016).
Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array.
Nat Biotechnol - Zetsche 2016 Cpf1 Multiplex
Kleinstiver et al. (2019).
Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.
Nat Biotechnol - Kleinstiver 2019 enCas12a
