Part:BBa_K3201000

Usage and Biology

Cpf1 is a type II clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided DNA-endonuclease from bacteria, such as Acidaminococcus sp. (AsCpf1), Francisella novicida (FnCpf1), and others (e.g. MbCpf1, LbCpf1,). AsCpf1 is a 1307 amino acid protein and its crystal structure has been resolved (1). Cpf1 uses a short crRNA, which guides Cpf1 to the crRNA-complementary DNA region.

The RuvC and Nuc domains carry out the hydrolysis of the phosphodiester bonds and thus DNA cleavage. However, it is possible to inactivate the Nuc domain and transform AsCpf1 into a nickase (nCpf1) that cleaves only the non-target strand and therefore leaves the double-stranded DNA nicked. The presence of the protospacer adjacent motif (PAM) is required for Cpf1 (and nCpf1) to cleave the DNA. AsCpf1 (and other Cpf1 proteins from other species) recognise the 5’-TTTV-3’ palindrome associated motif (PAM), where V = A, G, or C. Without the PAM there is little to none DNA cleavage even if the crRNA fully base pairs with the target DNA region.

References

(1) Stella, Stefano et al. "Structure Of The Cpf1 Endonuclease R-Loop Complex After Target DNA Cleavage". Nature, vol 546, no. 7659, 2017, pp. 559-563. Springer Science And Business Media LLC, doi:10.1038/nature22398.