Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a

Abstract

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

Keywords: CRISPR-Cas; Cas12a; Cas9; Cpf1; DNA cleavage; DNase; PAM; genetic engineering; genome editing; ssDNase.

Figure 1. Target ssDNA binding allosterically activates the RuvC catalytic site in Cas12a

(A) Schematic representation of the FnCas12a-mediated trans-cleavage of M13 DNA experiment. (B) Left and Right: FnCas12a-crRNA complexes were incubated with or without (non-)complementary ssDNA (ss) or dsDNA (ds) targets and non-target circular M13 ssDNA (left panel) or circular M13 dsDNA (right panel) substrates. Degradation products were resolved by agarose gel electrophoresis. M: 1kb DNA ladder marker, Comp.: complementary, ss: single-stranded, ds: double-stranded. (C) Top: Schematic diagram of the domain organization of FnCas12a. REC, recognition; PI, protospacer adjacent motif (PAM) interacting; WED, wedge; BH, bridge helix; Nuc, nuclease. Both the WED and RuvC domains are formed by three discontinuous segments of the protein sequence. Bottom: Sequence of the crRNA guide and TS DNA (see also Table S1) in the structure of the FnCas12a-crRNA-TS complex (see also panel D). Structurally disordered crRNA nucleotides are colored gray. (D) Overall structure of the FnCas12a-crRNA-TS complex. Domains are colored according to the scheme in panel C. For details of FnCas12a-crRNA-TS binding interactions, see Figure S1. (E) Surface representation of the binary FnCas12a-crRNA (PDB: 5NG6) and the ternary FnCas12a-crRNA-TS complex structures. Domains are colored according to the scheme in panel C. See Figure S2 for additional structural comparisons. (F) DNA substrates modeled into the activated catalytic site of FnCas12a. Left: A single stranded target DNA modeled in the RuvC catalytic site of FnCas12a (PDB: 5NFV). The modeled DNA is based on the structure of AacCas12b-crRNA bound to a DNA target (PDB: 5U33). Right: A double stranded DNA (PDB: 1BNA) modeled in the RuvC catalytic site of FnCas12a (PDB: 5NFV). Nucleotides colored yellow clash with the FnCas12a RuvC domain.

Figure 2. PAM recognition facilitates target dsDNA unwinding

(A) Schematic representation of the Fluorophore-Quencher (FQ)-reporter based trans-cleavage experiment. Briefly, dsDNA target binding by FnCas12a-crRNA complexes triggers trans-cleavage of non-target FQ-reporter DNA substrate, which allows subsequent detection of released fluorophores (F) that are no longer quenched by the quencher (Q). (B) Target dsDNA containing a 5’-YTA-3’ PAM trigger FnCas12a trans-cleavage of a non-target FQ-reporter. Left: schematic representation of ssDNA and dsDNA targets used in the experiments. Right: FnCas12a-crRNA complexes were incubated with dsDNA targets and a non-target FQ-reporter. Fluorescence (A.U.: arbitrary units) was measured over time. Data points represent the average of three replicates and error bars indicate standard deviation. For a FQ-reporter-based experiment with trans-activating DNAs with another sequence, see Figure S3. (C) The PAM is dispensable for trans-cleavage or pre-unwound dsDNA targets. Left: schematic representation of ssDNA and dsDNA targets used in the experiments. Right: FnCas12a-crRNA complexes were incubated with dsDNA targets and a non-target FQ-reporter. Fluorescence (A.U.: arbitrary units) was measured over time. Data points represent the average of three replicates and error bars indicate standard deviation. (D) The PAM is dispensable for cis-cleavage pf pre-unwound dsDNA targets. FnCas12a-crRNA complexes were incubated with dsDNA targets of which the TS 5’ end was Cy5-labeled. Cleavage products were resolved by denaturing (7 M Urea) 20% polyacrylamide gel electrophoresis.

Figure 3. Surface electrostatics of the REC lobe orchestrates NTS cleavage

(A) Sequences of the crRNA guide and TS and NTS DNA (see also Table S1) in the structure of the FnCas12a-crRNA-dsDNA complex. Structurally disordered nucleotides are colored gray. (B) Overall structure of the FnCas12a-crRNA-dsDNA complex. Protein domains are colored according to the scheme in Figure 1B, nucleic acids are colored according to the scheme in panel A. For details of FnCas12a-crRNA-TS binding interactions, see Figure S4. (C) Surface electrostatic potential map of the FnCas12a NUC lobe reveals the NTS-binding groove. The NTS is colored yellow and the REC lobe is omitted for clarity. Blue, positively charged region; red, negatively charged region. The inset panel displays the positively charged residues involved in NTS coordination. For a sequence alignment displaying the conservation of NTS residues see Figure S5A. For structural conservation of the NTS-binding groove see Figure S5B. (D) Mutation of NTS-binding groove residues lowers cis-cleavage efficiencies. FnCas12a, FnCas12a^{K1218A-K1287A-K1288}, and FnCas12a^{R1014A-R016A-K1066A-K1069A} were incubated with a crRNA and a target dsDNA (of which the TS was 5’-Cy5 labeled). Cleavage products were resolved by denaturing (7 M Urea) 20% polyacrylamide gel electrophoresis. Data points represent the average of duplicates and error bars indicate standard deviation. The inset shows the initial reaction curves.

Figure 4. Non-target DNA strand cleavage precedes target DNA strand cleavage

(A) Schematic representation of the sequences of TS and NTS DNA used for the experiments in panel B-D. Nucleotides colored red indicate nucleotides that are linked by phosphorothioates in modified TS and NTS strands. For Electromobility Shift Assays that demonstrate that phosphorothioate modifications do not hamper binding by FnCas12a-crRNA complexes, see Figure S6. (B-C) FnCas12a cleaves the NTS and TS sequentially. FnCas12a-crRNA complexes were incubated with dsDNA targets (of which the TS was 5’-Cy5 labeled) and cleavage products were resolved by denaturing (7 M Urea) 20% polyacrylamide gel electrophoresis. Data points in panel C represent the average of duplicates and error bars indicate standard deviation. (D) FnCas12a cleaves the TS in a pre-unwound dsDNA target, even if the NTS carries phosphorothioate modifications. FnCas12a-crRNA complexes were incubated with dsDNA targets (of which the TS was 5’-Cy5 labeled) and cleavage products were resolved by denaturing (7 M Urea) 20% polyacrylamide gel electrophoresis. Data points represent the average of duplicates and error bars indicate standard deviation.

Figure 5. Cas12a-crRNA releases PAM-distal DNA and remains bound to the PAM-proximal cleavage product

(A-D) FnCas12a-crRNA complexes were incubated with fluorophore-labeled dsDNA targets and analyzed by fluorescence-detection size exclusion chromatography. Elution fractions were further analyzed for protein and fluorophore-labeled nucleic acid content by SDS-PAGE and 20% denaturing (7 M Urea) polyacrylamide gel electrophoresis, respectively. In the schematic representation of the nucleic acids (top of each panel), the gray asterisk indicates the position of the fluorophore label and the red triangles indicate predicted TS (blue) and NTS (black) cleavage sites. IN: HPLC input; E1-6: Elution fractions; ctrl: Control sample containing catalytic mutant FnCas12a^{E1006Q-R1218A} instead of FnCas12a. All HPLC chromatograms including controls are shown in Figure S7. (E) Schematic representation of ssDNA and dsDNA targets used in the experiments, and the (predicted) cis-cleavage products of these targets that remains bound to FnCas12a. (F) Release of the PAM-distal segment of the cis-cleaved DNA targets facilitates trans-cleavage of non-target DNA. FnCas12a-crRNA complexes were incubated with ssDNA and dsDNA targets and a non-target FQ-reporter. Fluorescence (A.U.: arbitrary units) was measured over time. Data points represent the average of three replicates and error bars indicate standard deviation.

Figure 6. Schematic model of Cas12a-mediated cis- and trans-cleavage of DNA

Cas12a processes the 5’ end of its own crRNA guide and pre-orders nucleotides 1–5 of the crRNA seed segment. Canonical dsDNA target recognition initiates with PAM recognition by the WED and PI domains and promotes dsDNA target unwinding. R-loop formation initiates with the TS binding the pre-ordered crRNA seed segment. Processive crRNA-TS hybridization and simultaneous TS-NTS unwinding result in formation of the complete R-loop. Formation of the crRNA-TS heteroduplex induces conformational changes in the REC lobe resulting in allosteric unblocking of the catalytic site in the RuvC domain. The NTS binding groove guides the displaced NTS toward the RuvC catalytic site, resulting in cis-cleavage of the NTS. Subsequently, further unwinding of the PAM-distal TS-NTS duplex allows the TS to enter the RuvC catalytic site, resulting in cis-cleavage of the TS. The PAM-distal dsDNA is released, while the PAM-proximal dsDNA remains bound to the Cas12a-crRNA complex. This maintains Cas12a in a catalytically activated conformation, which permits trans-cleavage of non-target single-stranded DNAs.