NEW YORK (GenomeWeb) – The Broad Institute's Feng Zhang and his colleagues have characterized a new CRISPR enzyme, called Cpf1, that may enable more precise genome editing.
As Zhang and his colleagues described in Cell today, Cpf1 differs in some key ways from the commonly used Cas9 enzyme. It is a single RNA-guided endonuclease, it cuts the target site to leave sticky ends, and its T-rich protospacer-adjacent motif (PAM) provides greater flexibility in choosing those target sites. These characteristics, the researchers said, could be harnessed for more accurate genome editing.
"This has dramatic potential to advance genetic engineering," Eric Lander, the director of the Broad Institute, said in a statement. "The paper not only reveals the function of a previously uncharacterized CRISPR system, but also shows that Cpf1 can be harnessed for human genome editing and has remarkable and powerful features. The Cpf1 system represents a new generation of genome editing technology."
After searching through a catalog of CRISPR systems, Zhang and his colleagues focused on the Cpf1-containing CRISPR/Cas loci as a potential CRISPR system. Two candidate enzymes from Acidominococcus and Lachnospiraceae held the most promise as efficient genome-editing tools, they reported.
While the Cpf1 protein exhibits some similarities to Cas9 — it is predicted to have a RuvC-like endonuclease domain distantly related to that of Cas9 — it also shows some intriguing differences.
For instance, Cpf1 lacks the second HNH endonuclease harbored by Cas9 and has a mixed α/β N-terminal structure, in contrast to the α-helical recognition lobe in Cas9.
Cpf1 also harbors a PAM sequence — a requirement for DNA interference — that is distinct from that of Cas9. In Francisella novicida, Cpf1 has a 5' TTN PAM sequence, with the middle T of that recognition sequence apparently more critical than the others.
This T-rich PAM sequence could enable, the researchers added, genome editing in organisms with particular AT-rich genomes or into areas that are AT enriched, in contrast to the G-rich PAM sequence of Cas9.
Cpf1 may also represent a simpler genome-editing system, Zhang and his colleagues said.
Cpf1-based CRISPR loci process the CRISPR array into short mature crRNA that are about 43 nucleotides in length. Each of those crRNAs start with 19 nucleotides of the direct repeat, followed by the spacer sequence.
But the researchers were unable to identify any small RNA transcripts that could correspond to tracrRNAs.
This suggests that, in contrast to Cas9, Cpf1 is a single crRNA-guided endonuclease.
While Cas9 requires tracrRNA to process crRNA arrays and both crRNA and tracrRNA to mediate interference, Cpf1 likely processes crRNA arrays without tracrRNA, and Cpf1-crRNA complexes can alone cleave targeted DNA molecules.
This, Zhang and his colleagues said, could simplify both the design and delivery of genome-editing tools.
They noted that the shorter crRNA used by Cpf1 may be more practical to use as it's easier and cheaper to synthesize them than the 100 nucleotide crRNAs of the Cas9-based system.
Further, Cpf1 cleavage leaves a 5' overhang, which they said could offer an advantage over the blunt cut made by Cas9.
Blunt ends, the researchers noted, are subject to mutations as they are rejoined. And, at the same time, sticky ends could enable researchers to design a DNA insert so that it integrates into the genome in a certain orientation. It also offers a way to introduce DNA into the genome through non-homology-directed repair mechanisms, the researchers noted, and could enable editing of non-dividing cells.
Further, Cpf1 cleaves away from its recognition site, the researchers noted. In this system, even if the targeted gene is cleaved, researchers could still target it again and again.
"The unexpected properties of Cpf1 and more precise editing open the door to all sorts of applications, including in cancer research," noted the Broad's Levi Garraway, who was not involved in the study, in a statement.