CRISPR-associated protein 12a (Cas12a), formerly CRISPR from Prevotella and Francisella 1 (Cpf1)

Characteristics

Cleaves both DNA strands using a single RuvC-like nuclease domain, producing a staggered double-strand break with a 5' overhang. In FnCas12a the non-target strand is cut after the 18th protospacer base and the target strand after the 23rd, a 5-nucleotide overhang at the PAM-distal end; AsCas12a and LbCas12a cut the same way. The mature crRNA is 42-44 nt, 19 nt of direct repeat plus 23-25 nt of spacer, and Cas12a processes its own CRISPR array without a tracrRNA. Efficient cleavage requires at least 18 nt of spacer, with the seed in the first 5 nt at the spacer 5' end. Mutating the RuvC catalytic residues abolishes cleavage of both strands rather than producing a nickase. Digenome-seq maps 6 cleavage sites per crRNA for LbCas12a and 12 for AsCas12a, against over 90 for Cas9.

Applications: Indel formation in HEK293FT cells at efficiencies comparable to SpCas9, and multiplexed editing of up to four genes in mammalian cells and three in the mouse brain from a single crRNA array. Arrays of up to 25 spacers on one Pol II transcript combine editing with transcriptional activation or repression. LbCas12a ribonucleoprotein supports mutagenesis and homology-directed repair in zebrafish and Xenopus embryos.

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; the variants enAsCas12a and impLbCas12a widen this range. Temperature modulates activity, and AsCas12a works poorly in ectothermic organisms such as zebrafish and Xenopus. Target binding triggers indiscriminate trans cleavage of single-stranded DNA.

References

1. Zetsche et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell - Zetsche 2015 Cas12a
2. 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
3. Tóth et al. (2020). Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Res - Tóth 2020 LbCas12a
4. Moreno-Mateos et al. (2017). CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun - Moreno-Mateos 2017 Cpf1 Temperature
5. Zetsche et al. (2016). Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat Biotechnol - Zetsche 2016 Cpf1 Multiplex
6. Campa et al. (2019). Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods - Campa 2019 SiT-Cas12a