Genetic Engineering Publications - GEG Tech top picks

Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here the scientists provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

Over the past few years, intensive studies have provided an atomic view of the crRNA-guided target interference mechanisms in different types of CRISPR-Cas systems. Here, the authors review the recent progress toward structural and mechanistic understanding of the diverse crRNP effector nucleases.

Cpf1 is a CRISPR−Cas endonuclease that, as a genome engineering tool, may have several advantages over Cas9, such as the production of staggered cuts with overhangs rather than blunt end cuts. Both Cpf1 and Cas9 use a CRISPR RNA (crRNA) to recognize target DNA, which is then cleaved using a RuvC nuclease domain. However, target cleavage by Cas9 additionally requires a HNH nuclease domain, whereas a second nuclease domain had not been identified for Cpf1. Yamano et al. and Dong et al. report crystal structures of Cpf1 that reveal a second putative nuclease domain, with a novel fold and no detectable homologues. Using mutational analysis, Yamano et al. show that this domain cleaves the target DNA at a distal site to produce the staggered cut. Remarkably, Fonfara et al. found that, in addition to DNase domains, Cpf1 has an RNase domain, which processes precursor transcripts into crRNAs. Thus, Cpf1 is an all-in-one machine for enacting CRISPR−Cas immunity.

Here the scientists show that Lachnospiraceae bacterium (Lb) and Acidaminococus sp. (As) Cpf1 orthologs have RNase activities that can excise multiple CRISPR RNAs (crRNAs) from a single RNA polymerase II–driven RNA transcript expressed in mammalian cells. This property simplifies modification of multiple genomic targets and can be used to increase the efficiency of Cpf1-mediated editing.

In this study, the scientists deployed Cpf1 to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

Here, the scientists report that four to six bases at the 3′ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, they were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.

This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.