The CRISPR gene-editing toolbox may soon make room for a newly characterized DNA-snipping enzyme. The new enzyme, Cpf1, is a class II CRISPR endonuclease, like the well-known Cas9. Yet Cpf1 has features that are distinct from Cas9. Indeed, these features suggest that genetic editing via CRISPR-Cpf1 could be even more flexible and powerful than genetic editing via CRISPR-Cas9.
Two Cpf1 enzymes emerged as particularly promising gene-editing candidates from a search that encompassed hundreds of CRISPR systems. One of the enzymes is from Acidaminococcus bacteria; the other, from Lachnospiraceae bacteria. Both enzymes are capable of targeting genomic loci in human cells.
These findings, which were established by researchers centered at the Broad Institute, were presented September 25 in the journal Cell, in an article entitled, “Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.” The article summarized Cpf1’s distinct features as follows: “Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break.”
The first of these features—the need for just one RNA in a gene-editing complex—means that Cpf1 could be simpler to use than Cas9, which in its natural form requires two RNAs. Also, Cpf1 is smaller than the standard Cas9, making it easier to deliver into cells and tissues.
The second feature is also significant. Because it targets protospacer-adjacent motif (PAM) sequences distinct from those targeted by Cas9, Cpf1 may be able to cut DNA at locations Cas9 misses. Cpf1 could even be advantaged in targeting certain genomes, such as those in the malaria parasite or in humans. In addition, Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be recut, allowing multiple opportunities for correct editing to occur.
The third and final feature may be the most important. Essentially, Cpf1 cuts DNA in a different manner than Cas9. According to the authors of the Cell article, “Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5′ T-rich PAM.”
Whereas Cas9 acts like a conventional pair of scissors, cutting straight across paired DNA strands, Cpf1 acts more like a pair pinking shears, applying staggered cuts. When Cpf1 cuts DNA, the cut ends on either DNA strand are offset a bit, leaving short overhangs. These staggered ends are less vulnerable to mutations than the “blunt ends” that are left by Cas9. Consequently, CRISPR-Cpf1-altered genomes may be less prone to fraying during tailoring.
The identification and characterization of Cpf1 was led by Feng Zhang, who harnessed the CRISPR-Cas9 system for mammalian gene editing in 2013, independently of George Church at Harvard, who carried out similar work on Cas9. With respect to the new work, which indicates that Cpf1 is capable of efficient genome-editing activity in human cells, Zhang said, “We were thrilled to discover completely different CRISPR enzymes that can be harnessed for advancing research and human health.”
The Broad Institute and MIT plan to offer nonexclusive licenses to enable commercial tool and service providers to add this enzyme to their CRISPR pipeline and services, further ensuring availability of this new enzyme to empower research. These groups plan to offer licenses that best support rapid and safe development for appropriate and important therapeutic uses.
“We are committed to making the CRISPR-Cpf1 technology widely accessible,” Zhang explained. “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”