NEW YORK (GenomeWeb) – A new study from CRISPR/Cas9 pioneer Emmanuelle Charpentier suggests that CRISPR/Cpf1, a new system first described in 2015, can cleave RNA in addition to DNA.
Published today in Nature, the study suggests Cpf1 from Francisella novicida is a dual nuclease. The authors said it is the most simplistic CRISPR system discovered so far, using only one enzyme to process its guide RNA and using that same enzyme and guide RNA to target and cleave invading DNA.
One reason CRISPR/Cpf1 is notable among CRISPR systems is that in its natural setting, it requires only one CRISPR RNA (crRNA) to target and cleave DNA. CRISPR/Cas9 technically requires a crRNA complementary to the target and a tracrRNA — which both hybridizes to the crRNA and interacts with the Cas9 enzyme — to work; however, in genome editing applications, the two RNAs are often fused into a chimeric single guide RNA.
Charpentier, a director at the Max Planck Institute for Infection Biology in Berlin, and her co-authors suggest that in F. novicida, Cpf1 processes the crRNA prior to maturation, a point after which the crRNA complexes with the enzyme and enables genomic cleavage. Specifically, the enzyme lops off approximately 19 nucleotides using a specific RNA-cleaving domain.
"Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs," the authors wrote.
The RNA- and DNA-cleaving domains are separate, since the DNA motif show cleavage activity "only against double-stranded and single-stranded target DNA, but no activity against single-stranded RNA, double-stranded RNA or RNA–DNA heteroduplexes," they wrote.
When Feng Zhang of the Broad Institute and the Massachusetts Institute of Technology and Eugene Koonin of the National Center for Biotechnology Information first described the CRISPR/Cpf1 system, they revealed an entirely new branch of the CRISPR family tree. The well-known CRISPR/Cas9 is just one of five known "types" of bacterial immune systems, which can involve multiple enzymes. Though the chimeric single guide RNA in artificial Cas9-based gene editing systems obviates it, in nature the Cas9 system needs an RNase III to process the crRNA and tracrRNA prior to targeting and cleaving DNA.
But Cpf1 appeared to be simpler, and the new study suggests it can perform its own crRNA processing. In addition to demonstrating the dual-nuclease nature of Cpf1, the researchers expanded the protospacer-adjacent motif characterization.
While Zhang and Koonin had shown Cpf1 to recognize a PAM of TTN upstream of the cleavage site, Charpentier's team expanded that to YTN, where Y is any pyrimidine.
They also showed that the Cpf1 cleaves DNA using either magnesium or calcium ions, but noted that "the physiological relevance of Cpf1 using both ions for DNA cleavage remains undetermined and requires further investigation."