Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system

Abstract

The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. 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. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.

Figure 1. The Francisella tularensis subsp. novicida U112 Cpf1 CRISPR locus provides immunity against transformation of plasmids containing protospacers flanked by a 5′-TTN PAM

(A) Organization of two CRISPR loci found in Francisella tularensis subsp. novicida U112 (NC_008601). The domain architectures of FnCas9 and FnCpf1 are compared. (B) Schematic illustrating the plasmid depletion assay for discovering the PAM position and identity. Competent E. coli harboring either the heterologous FnCpf1 locus plasmid (pFnCpf1) or the empty vector control were transformed with a library of plasmids containing the matching protospacer flanked by randomized 5′ or 3′ PAM sequences and selected with antibiotic to deplete plasmids carrying successfully-targeted PAM. Plasmids from surviving colonies were extracted and sequenced to determine depleted PAM sequences. (C) Sequence logo for the FnCpf1 PAM as determined by the plasmid depletion assay. Letter height at each position is measured by information content; error bars show 95% Bayesian confidence interval. (D) E. coli harboring pFnCpf1 provides robust interference against plasmids carrying 5′-TTN PAMs (n = 3, error bars represent mean ± S.E.M.). See also Figure S1.

Figure 2. Heterologous expression of FnCpf1 and CRISPR array in E. coli is sufficient to mediate plasmid DNA interference and crRNA maturation

(A) Small RNA-seq of Francisella tularensis subsp. novicida U112 reveals transcription and processing of the FnCpf1 CRISPR array. The mature crRNA begins with a 19 nt partial direct repeat followed by 23–25 nt of spacer sequence. (B) Small RNA-seq of E. coli transformed with a plasmid carrying synthetic promoter-driven FnCpf1 and CRISPR array shows crRNA processing independent of Cas genes and other sequence elements in the FnCpf1 locus. (C) E. coli harboring different truncations of the FnCpf1 CRISPR locus shows that only FnCpf1 and the CRISPR array are required for plasmid DNA interference (n = 3, error bars show mean ± S.E.M.).

Figure 3. FnCpf1 is guided by crRNA to cleave DNA in vitro

(A) Schematic of the FnCpf1 crRNA-DNA targeting complex. Cleavage sites are indicated by red arrows. (B) FnCpf1 and crRNA alone mediated RNA-guided cleavage of target DNA in a crRNA- and Mg²⁺-dependent manner. (C) FnCpf1 cleaves both linear and supercoiled DNA. (D) Sanger sequencing traces from FnCpf1-digested target show staggered overhangs. The non-templated addition of an additional adenine, denoted as N, is an artifact of the polymerase used in sequencing (Clark, 1988). Reverse primer read represented as reverse complement to aid visualization. See also Figure S3. (E) Dependency of cleavage on base-pairing at the 5′ PAM. FnCpf1 can only recognize the PAM in correctly Watson-Crick paired DNA. See also Figures S2 and S3.

Figure 4. Catalytic residues in the C-terminal RuvC domain of FnCpf1 are required for DNA cleavage

(A) Domain structure of FnCpf1 with RuvC catalytic residues highlighted. The catalytic residues were identified based on sequence homology to Thermus thermophilus RuvC (PDB ID: 4EP5). (B) Native TBE PAGE gel showing that mutation of the RuvC catalytic residues of FnCpf1 (D917A and E1006A) and mutation of the RuvC (D10A) catalytic residue of SpCas9 prevents double stranded DNA cleavage. Denaturing TBE-Urea PAGE gel showing that mutation of the RuvC catalytic residues of FnCpf1 (D917A and E1006A) prevents DNA nicking activity, whereas mutation of the RuvC (D10A) catalytic residue of SpCas9 results in nicking of the target site. See also Figure S4.

Figure 5. crRNA requirements for FnCpf1 nuclease activity in vitro

(A) Effect of spacer length on FnCpf1 cleavage activity. (B) Effect of crRNA-target DNA mismatch on FnCpf1 cleavage activity. See also Figure S3E. (C) Effect of direct repeat length on FnCpf1 cleavage activity. (D) FnCpf1 cleavage activity depends on secondary structure in the stem of the direct repeat RNA structure. (E) FnCpf1 cleavage activity is unaffected by loop mutations but is sensitive to mutation in the 3′-most base of the direct repeat. See also Figure S4.

Figure 6. Analysis of Cpf1-family protein diversity and function

(A) Phylogenetic tree of 16 Cpf1 orthologs selected for functional analysis. Conserved sequences are shown in dark gray. The RuvC domain, bridge helix, and zinc finger are highlighted. (B) Alignment of direct repeats from the 16 Cpf1-family proteins. Sequences that are removed post crRNA maturation are colored gray. Non-conserved bases are colored red. The stem duplex is highlighted in gray. (C) RNAfold (Lorenz et al., 2011) prediction of the direct repeat sequence in the mature crRNA. Predictions for FnCpf1 along with three diverged type V loci are shown. (D) Type V crRNAs from different bacteria with similar direct repeat sequences are able to function with FnCpf1 to mediate target DNA cleavage. (E) PAM sequences for 8 Cpf1-family proteins identified using in vitro cleavage of a plasmid library containing randomized PAMs flanking the protospacer. See also Figures S5 and S6.

Figure 7. Cpf1 mediates robust genome editing in human cell lines

(A) Eight Cpf1-family proteins are individually expressed in HEK 293FT cells using CMV-driven expression vectors. The corresponding crRNA is expressed using a PCR fragment containing a U6 promoter fused to the crRNA sequence. Transfected cells were analyzed using either Surveyor nuclease assay or targeted deep sequencing. (B) Schematic showing the sequence of DNMT1-targeting crRNA 3. Sequencing reads show representative indels. (C) Comparison of in vitro and in vivo cleavage activity. The DNMT1 target region was PCR amplified and the genomic fragment was used to test Cpf1-mediated cleavage. All 8 Cpf1-family proteins showed DNA cleavage in vitro (top), but only candidates 7 – AsCpf1 and 13 – Lb3Cpf1 facilitated robust indel formation in human cells. (D) Cpf1 and SpCas9 target sequences in the human DNMT1 locus. (E) Comparison of Cpf1 and SpCas9 genome editing efficiency. Target sites correspond to sequences shown in Figure 7D. See also Figure S7.