Cpf1: A New Tool for CRISPR Genome Editing

Updated Aug 13, 2020.

Note: Cpf1 is also called Cas12a.

In 2015, Zetsche et al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher on CRISPR/Cas9. 

Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015.

How was Cpf1 found and tested?

Class 2 CRISPR systems, including the type II Cas9-based system, require a single-component nuclease to mediate cleavage rather than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was annotated in several genomes and is classified as a type V CRISPR system. Like Cas9, Cpf1 contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating that Cpf1 functions differently. Since Cpf1 loci are widely distributed across bacterial species, Zetsche et al. hypothesized that Cpf1 might represent a functional CRISPR nuclease that could be adapted for genome editing. The use of a different nuclease could potentially overcome some of Cas9’s shortcomings - namely its blunt double stranded cleavage and G-rich PAM requirement.

Zetsche et al. started from square one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid depletion assay to discover FnCpf1’s PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After sequencing and searching for cellular RNAs important for Cpf1 function, they found that mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat preceding the spacer rather than following it. The Cpf1 crRNA is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat region is necessary for cleavage of a target. Cpf1 also does not require an additional tracrRNA.

Once they had determined the minimal elements of CRISPR-Cpf1, Zetsche et al. turned to characterizing its cleavage pattern. Again, they were in for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With this information, they turned to a cell culture system to see if any Cpf1 nucleases would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found two that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and LbCpf1 (1307 and 1228 amino acids long respectively), both cleave in a staggered pattern similar to FnCpf1.

Potential advantages of Cpf1 over Cas9

Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems and the easiest to adapt to genome editing, but the introduction of type V Cpf1-driven systems has added another option to the CRISPR toolbox. Cpf1’s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning. Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-directed repair (HDR). Cpf1 also expands the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9.

Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these crRNAs should be significantly cheaper than that of the ~100 nt crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts, they will also be easier to deliver in low-capacity vectors, such as adeno-associated viral (AAV) vectors. In 2017, Zetsche et al. developed a Cpf1 multiplexing approach using a single crRNA array to express up to 4 crRNAs.

Zetsche et al. also suggest that Cpf1 may improve the frequency of HDR over non-homologous endjoining (NHEJ). Cas9-mediated NHEJ usually destroys the PAM site due to its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far away from the PAM, NHEJ might retain the PAM site. Therefore, if HDR does not initially occur after Cpf1-mediated cleavage, the continued presence of the PAM may give Cpf1 the ability to cleave again and possibly mediate HDR. This “second chance” mechanism might improve the frequency of desired HDR edits, but the possibility has not yet been experimentally confirmed. To prevent new editing post-HDR, repair templates should remove the PAM sequence.

Comparing Cpf1 and Cas9 on-target and off-target efficiency

When Cpf1 was first identified, we didn't know much about its on-target and off-target editing efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing efficiency for the Cpf1 orthologs was only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency varies widely with gRNA sequence.

Both groups used multiple methods to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches, but variably so, with Kleinstiver et al. reporting that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an essential function in Cpf1-mediated editing, as Kleinstiver et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence.

Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each graph. Figure from Kleinstiver et al., 2016.

Cpf1’s strength may lie in its low off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally predicted off-target sites, Cpf1 does not mediate detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. Kim et al. also compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs show lower off-target activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins (RNPs) failed to induce off-target editing in a cell culture model.

The application of Cpf1 to genome editing is exciting both in terms of basic science and translational applications. This discovery of this type V CRISPR system proved we had a lot more to learn about CRISPR biology. Later work, like the adaptation of Cas13 to RNA targeting and RNA editing, has further shown the diversity of CRISPR-based systems.

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References

1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227.

• Find plasmids from this publication at Addgene.

2. Zetsche, Bernd, et al. "Multiplex Gene Editing by CRISPR–Cpf1 Using a Single crRNA Array." Nat Biotechnol. 35.1 (2016): 31-34. PubMed PMID: 27918548.

• Find plasmids from this publication at Addgene.

3. Kim, Daesik, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. (2016). PubMed PMID: 27272384

• Find plasmids from this publication at Addgene.

Kleinstiver, Benjamin P., et al. "Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nat Biotechnol. (2016). PubMed PMID: 27347757.

• Find plasmids from this publication at Addgene.

Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas systems." Nat Rev Microbiol. (2015). PubMed PMID: 26411297.

Resources at Addgene

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Additional Resources

• Zhang lab Cpf1 blog post on Benchling