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. Author manuscript; available in PMC: 2019 Nov 15.
Published in final edited form as: Mol Cell. 2019 Jan 10;73(3):589–600.e4. doi: 10.1016/j.molcel.2018.11.021

Mechanistic Insights into the Cis- and Trans-acting Deoxyribonuclease Activities of Cas12a

Daan C Swarts 1, Martin Jinek 1,2
PMCID: PMC6858279  EMSID: EMS84915  PMID: 30639240

Summary

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 (ssDNA) in trans. To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that crRNA-target DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn, cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding 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 mechanism of both the cis- and trans-acting deoxyribonuclease activities of Cas12a enzymes, allowing their further exploitation as genome editing tools.

Keywords: CRISPR-Cas, Cas12a, Cpf1, Cas9, DNA cleavage, deoxyribonuclease, ssDNase, PAM, genome editing, genetic engineering

Introduction

In prokaryotes, CRISPR-Cas systems (clustered regularly interspaced palindromic repeats and CRISPR-associated proteins) function as programmable immune systems that utilize CRISPR RNAs (crRNAs) as guide molecules for the recognition and targeting of invasive nucleic acids such as viral DNA (Koonin et al., 2017; Mohanraju et al., 2016). The DNA targeting Class 2 CRISPR-Cas systems, which comprise types II and type V, elicit antiviral immunity by means of single multidomain nuclease enzymes that mediate crRNA-guided target DNA binding and catalyze target DNA cleavage. Cas9 and Cas12a proteins, the respective effector nucleases of type II and type V-A systems, mediate crRNA-guided DNA cleavage by recognizing two distinct sequence elements in the target DNA: (i) a ‘protospacer’ sequence, specified by Watson-Crick base-pairing interactions with the crRNA, and (ii) a protospacer adjacent motif (PAM), recognized via sequence- and/or shape-specific protein-DNA interactions (Swarts and Jinek, 2018). Upon binding of a cognate target DNA, the complementary spacer-derived segment of the crRNA and the protospacer ‘target strand’ (TS) form an RNA-DNA heteroduplex. At the same time, the non-target strand (NTS) of the protospacer is displaced. Formation of this so-called ‘R-loop’ structure catalytically activates both Cas9 and Cas12a, triggering cis-cleavage of both target DNA strands. The ability to program Cas9 and Cas12a with a crRNA sequence of choice has led to their repurposing for precision genome editing and for regulation of gene expression (Barrangou and Doudna, 2016; Swarts and Jinek, 2018; Wang et al., 2016; Wright et al., 2016).

Despite similar functionalities, Cas9 and Cas12a proteins have distinct evolutionary histories and therefore distinct mechanistic properties (Shmakov et al., 2017; Swarts and Jinek, 2018). Streptococcus pyogenes Cas9, which is widely used for genome editing, recognizes a 5’-NGG-3’ PAM directly downstream of the protospacer NTS (Anders et al., 2014). In contrast, Cas12a orthologues used for genome editing recognize a 5’-TTTV-3’ PAM directly upstream of the protospacer NTS (Gao et al., 2016; Sun et al., 2018; Świat et al., 2017; Tu et al., 2017; Yamano et al., 2016; Zetsche et al., 2015). However, Cas12a can also target DNAs with ‘suboptimal’ PAMs (5’-TTV-3’, 5’-TCTV-3’, 5’-TCCV-3’, and 5’-CCCV-3’), albeit often at lower efficiencies (Gao et al., 2017; Nishimasu et al., 2017; Tu et al., 2017; Yamano et al., 2017). Cas9 and Cas12a furthermore use different mechanisms for DNA cleavage. Cas9 cleaves the TS and NTS with its HNH and RuvC domains, respectively (Gasiunas et al., 2012; Jinek et al., 2012). This typically results in PAM-proximal double-strand DNA breaks (DSBs) with blunt-ends or 1-nucleotide overhangs. In contrast, Cas12a uses a single RuvC nuclease domain for cleavage of both strands of the target DNA (Swarts et al., 2017), generating PAM-distal DSBs with larger (5-7 nt) 5’ overhangs (Zetsche et al., 2015). While both Cas9 and Cas12a initially cleave a target dsDNA at a single site, both enzymes catalyze trimming of the cleaved target ends in vitro (Jinek et al., 2012; Stephenson et al., 2018), yielding heterogeneous cleavage products. Finally, Cas12a exhibits two distinct DNA cleavage modes (Chen et al., 2018; Li et al., 2018). Besides cleaving dsDNA targets in a crRNA-guided manner in cis, Cas12a also possesses sequence-independent deoxyribonuclease activity that cleaves non-target single-stranded DNA in trans (Chen et al., 2018; Li et al., 2018). The trans-acting nuclease activity is triggered by hybridization of the crRNA to a complementary target DNA strand (Chen et al., 2018; Li et al., 2018).

Structural studies of Cas12a have revealed its overall molecular architecture and mechanism of target DNA recognition. The enzyme has a bilobed architecture in which the N-terminal REC lobe, consisting of the α-helical domains REC1 and REC2, is connected by the Wedge (WED) domain to the C-terminal NUC lobe comprising the PAM interacting (PI), Bridge Helix (BH), RuvC and Nuc dmains (Dong et al., 2016; Gao et al., 2016; Stella et al., 2017; Swarts et al., 2017; Yamano et al., 2016). The crRNA is recognized via sequence- and shape-specific interactions with the repeat-derived part of the crRNA, which forms a pseudoknot structure (Dong et al., 2016; Fonfara et al., 2016). crRNA binding results in structural pre-ordering of the seed segment in the crRNA, thereby priming the Cas12-crRNA complex for target DNA recognition (Swarts et al., 2017). Shape- and sequence-specific recognition of the PAM then facilitates target dsDNA binding and is prerequisite for catalyzing DNA cleavage (Gao et al., 2016; Yamano et al., 2016; Zetsche et al., 2015). Despite these insights, the molecular mechanisms underpinning cis- and trans-acting deoxyribonuclease activities of Cas12a have remained unclear.

To obtain insights into both cis- and trans-acting DNA cleavage activities of Cas12a, we have determined the crystal structures of Francisella novicida U112 Cas12a (FnCas12a) in ternary complex with a crRNA and either ssDNA or dsDNA targets. Together with corroborating biochemical experiments, the structure of Cas12a bound to a guide crRNA and single-stranded DNA target reveals that crRNA-TS DNA duplex formation, rather than PAM binding, drives a conformational rearrangement that allosterically activates the Cas12a RuvC domain. Although PAM binding does not play a role in the catalytic activation of Cas12a, it is essential for the recognition and cleavage of dsDNA targets since it promotes dsDNA strand separation and consequently R-loop formation. We have additionally determined a crystal structure of Cas12a-crRNA in complex with a dsDNA target, revealing that the NTS of the target DNA is electrostatically guided toward the catalytic site in the RuvC domain, thus facilitating sequential cleavage of the NTS and TS DNA strands. We furthermore demonstrate that after target DNA cleavage by Cas12a, the PAM proximal-DNA cleavage product remains bound to the Cas12a-crRNA complex, which maintains Cas12a in a catalytically activated state and allows for degradation of non-target single stranded DNA in trans.

Results

Target ssDNA binding allosterically activates the RuvC catalytic site in Cas12a

In addition to cleaving target dsDNAs in cis, Cas12a enzymes were recently reported to possess trans DNA cleavage activity triggered by target ssDNA binding (Chen et al., 2018; Li et al., 2018). To corroborate these findings, we programmed Francisella novicida Cas12a (FnCas12a) with a crRNA and incubated the resulting complex with circular ssDNA (M13 ssDNA) or circular dsDNA (M13 dsDNA) substrates that bear no complementarity to the crRNA. Next, we added to the reaction 20-nt ssDNA oligonuclotides that lacked a PAM and were either fully complementary or lacked complementarity to the spacer-derived segment of the crRNA (Figure 1A). In the presence of a non-complementary ssDNA, FnCas12a did not degrade the M13 DNAs. In contrast, the presence of a complementary target ssDNA caused complete FnCas12a-mediated degradation of M13 ssDNA, but not M13 dsDNA (Figure 1B). This activity was not observed for the RuvC catalytic site mutant FnCas12aE1006Q, indicating that the observed in trans-cleavage of ssDNA substrates is mediated by the RuvC catalytic site that also mediates canonical cis-cleavage of target dsDNA. These findings are in agreement with the previously reported trans-acting activity of Cas12a (Chen et al., 2018; Li et al., 2018) and, as a PAM is absent in the trans-activating ssDNA, suggest that crRNA-TS hybridization alone drives allosteric activation of Cas12a.

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

Figure 1

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(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.

To obtain structural insights into the allosteric mechanism underpinning catalytic activation of Cas12a, we determined the crystal structure of a ternary complex consisting of FnCas12a bound to a crRNA guide and a 20-nt PAM-less target ssDNA (FnCas12a-crRNA-ssDNA complex, Figures 1C-D and S1) at a resolution of 2.98 Å (Table 1). In agreement with other Cas12a structures, the FnCas12a-crRNA-ssDNA complex maintains a bilobed architecture. The first 20 nucleotides of the spacer-derived segment of the crRNA (A1-U20) form Watson-Crick base pairs with the TS, while the 3’-terminal nucleotides of the crRNA (U22-G24) are structurally disordered. Previously determined structures of binary Cas12a-crRNA complexes (PDB: 5NG6 and 5ID6) showed that the REC2 domain occludes the catalytic site in the RuvC domain (Figure 1E), which likely prevents not only cis-cleavage of target dsDNA, but also trans-cleavage of non-target ssDNAs. This is consistent with the observed lack of trans-acting nuclease activity in the absence of a trans-activating target DNA (Figure 1B).

Table 1. Crystallographic Data Collection and Refinement Statistics.

Dataset TS-only complex Ternary R-loop complex
FnCas12a WT E1006Q
RNA crRNA1 crRNA1
DNA oDS311 (TS) oDS142 (TS) + oDS141 (NTS)
X-ray source SLS PXIII SLS PXIII
Space group P21 21 21 P1 21 1
Cell dimensions
  a, b, c (Å) 78.80, 188.58, 284.12 82.17, 141.68, 89.48
  α, β, γ, (°) 90, 90, 90 90, 97.43, 90
Wavelength (Å) 1 1.008
Resolution (Å)* 48.67 – 2.98 (3.09 – 2.98) 47.61 – 2.65 (2.75 – 2.65)
Rmeas (%)* 18.09 (189.7) 8.18 (190.8)
CC1/2 (%) 0.999 (0.675) 1 (0.638)
I/σI* 18.87 (2.22) 17.86 (0.96)
Completeness (%)* 99.96 (100) 99.60 (98.67)
Multiplicity* 26.2 (27.6) 6.9 (6.8)
Refinement
Resolution (Å) 2.98 2.65
No. reflections 87,496 58,703
Rwork / Rfree 0.204 / 0.231 0.248 / 0.264
No. atoms
Protein 20,893 10,578
Nucleic acid 2476 2266
Ion/ligand 207 108
Water 52 30
B Factors
Mean 85.59 107.88
Protein 87.55 106.31
Nucleic acid 68.43 116.48
Ion/ligand 98.52 89.65
Water 63.65 80.29
RMSDs
Bond lengths (Å) 0.005 0.003
Bond angles (°) 0.68 0.62
Ramachandran plot
Favoured (%) 97.08 97.88
Allowed (%) 2.92 2.12
Outliers (%) 0.00 0.00
Molprobity
Clashscore 7.14 8.42
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*

Values in parentheses denote highest resolution shell

Although both ssDNA and dsDNA target binding induced cleavage of the M13 ssDNA substrate in trans, neither target DNA was able to trigger trans-cleavage of M13 dsDNA (Figure 1B). We previously modelled the binding of a ssDNA substrate in the RuvC catalytic site based on superpositions with the structures DNA-bound complexes of Cas12b (Swarts et al., 2017). In agreement with the trans-cleavage experiments, modeling of a dsDNA substrate reveals that the RuvC dsDNA substrate cannot accommodate a B-form DNA duplex (PDB: 1BNA) due to severe steric clashes of both dsDNA strands with the RuvC domain (Figure 1F). Consequently, this implies that cis-cleavage of a dsDNA target requires that the dsDNA is unwound to allow sequential cleavage of TS and NTS strands in the RuvC active site.

Structural superposition of the binary FnCas12a-crRNA (PDB: 5NG6) and ternary FnCas12a-crRNA-ssDNA complex structures reveals the conformational changes associated with catalytic activation by TS binding (Figures 1E and S2; root-mean-square deviation (RMSD): 12.54 Å). The NUC lobe is largely unperturbed by TS binding (RMSD of NUC lobe alignment: 2.61 Å), with only a small rotation of the Nuc domain by ~9° relative to the remainder of the NUC lobe. In contrast, the REC lobe undergoes a major conformational rearrangement (RMSD of REC lobe alignment: 15.26 Å). The REC1 domain rotates approximately 33° relative to the NUC lobe and thereby moves closer to the NUC lobe and to the crRNA-TS heteroduplex (Figure S2B), allowing formation of stabilizing interactions between the crRNA-TS heteroduplex and REC1 (Figure S1). The REC2 domain undergoes a rotation of 46° and a translation of ~9.5 Å relative to the REC1 domain. This is essential to accommodate the PAM-distal end of the crRNA-TS heteroduplex, which would otherwise result in severe clashes with the REC2 domain (Figure S1C). In addition, the rearrangement results in the formation of stabilizing interactions between the crRNA-TS heteroduplex and REC2 residues (Figure S1).

To investigate conformational differences between ternary structures bound to either ssDNA or dsDNA targets, we superimposed the structure of the FnCas12a-crRNA-TS complex and previously determined ternary structures of FnCas12a-crRNA complexes bound to intact (PDB: 5MGA) and cleaved (PDB: 5NFV) dsDNA targets (Figures S2C and S2D). The structure of the FnCas12a-crRNA-ssDNA complex is most similar to that of the cleaved dsDNA-bound complex (RMSD: 2.76 Å; Figure 2C). We observe minor narrowing of the PI domain in which the PAM interacting residues Lys613 and Lys671 move by ~1 Å and ~2.5 Å, respectively, toward the PAM of the dsDNA target. The intact dsDNA-bound complex displays a similar narrowing of the PI domain but additionally adopts a slightly more ‘opened’ conformation of the bilobed scaffold (RMSD: 4.51 Å; Figure 2D), in which the RuvC and Nuc domains have moved away from the REC lobe to accommodate the target dsDNA. The observation that the conformation of the FnCas12a-crRNA-ssDNA structure is similar to that of the two dsDNA-bound structures, corroborates biochemical experiments demonstrating that crRNA-TS hybridization alone is sufficient to allosterically activate FnCas12a. Collectively, the structural studies reveal that both ssDNA and dsDNA target binding results in the REC2 domain moving away from the NUC lobe, thus permitting substrate DNA access to the now exposed RuvC catalytic site.

Figure 2. PAM recognition facilitates target dsDNA unwinding.

Figure 2

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(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.

PAM recognition facilitates target dsDNA unwinding

The observation that target ssDNA binding is sufficient for allosteric activation of Cas12a implies that neither PAM recognition nor NTS coordination are per se required for the allosteric activation of the RuvC nuclease domain. However, the presence of a PAM in dsDNA targets is essential both during cis-cleavage of target dsDNA (Zetsche et al., 2015) and for dsDNA target-mediated trans-cleavage (Chen et al., 2018). Using a previously described fluorophore-quencher (FQ) reporter assay (Chen et al., 2018), we investigated the PAM requirement for dsDNA-induced trans-activation of FnCas12a (i.e. activation of non-target ssDNA cleavage in trans; Figure 2A). Trans-activation of FnCas12a was highest in the presence of a single stranded target DNA (TS), followed by dsDNA targets containing four (5’-TTTA-3’) or three (5’-TTA-3’) PAM base pairs (Figure 2B). dsDNA targets containing a 5’-CTA-3’ PAM also triggered trans-activation of FnCas12a, albeit at a slightly lower efficiency. These observations are in agreement with a recent study reporting that FnCas12a efficiently targets DNA with 5’-YTA-3’ PAMs (Tu et al., 2017). In contrast, almost no trans-cleavage was observed in the presence of dsDNA targets with shorter PAM motifs (Figure 2B). Similar results were observed for another set of dsDNA targets with different protospacer sequences (Figure S3). Thus, under the tested conditions, dsDNA-induced allosteric activation of Cas12a activity minimally required the presence of a 5’-YTV-3’ PAM.

Cas9 has been demonstrated to rely on PAM recognition to initiate unwinding of the protospacer segment in the dsDNA target, thus facilitating subsequent guide RNA invasion and R-loop formation (Anders et al., 2014; Jiang et al., 2016). As for Cas9, PAM recognition by FnCas12a induces a bend in the target DNA (Stella et al., 2017; Swarts et al., 2017), which could facilitate dsDNA unwinding and crRNA-TS hybridization. To test whether PAM binding is important for dsDNA unwinding in Cas12a, we extended our trans-cleavage FQ-reporter assays to include DNA targets that had canonical (5’-TTTA-3’) or mutated (5’-GCGT-3’) PAM sequences, and a 24-nt protospacer segment in which the TS and NTS were either fully duplexed (dsDNA) or unwound (unwDNA) (Figure 2C). These experiments indicate that, while the presence of a PAM is required for trans-activation by fully duplexed dsDNA targets, pre-unwound DNA targets induce trans-cleavage activity in a PAM-independent manner. Similarly, the PAM was dispensable for cis-cleavage of unwDNA (Figure 2D). It should be noted that compared to canonical cis-cleavage of dsDNA substrates, cis-cleavage of unwDNA substrate was less efficient and yielded smaller TS cleavage products. This can possibly be explained by the fact that the protospacer is unwound beyond the predicted cleavage location in the TS in the pre-unwound substrate (Stella et al., 2017; Zetsche et al., 2015), which could perturb the binding of the TS in the RuvC catalytic site and result in cleavage at a distinct site within the TS. Nevertheless, these findings corroborate observations that PAM-binding is dispensable for allosteric activation of trans-nuclease activity in Cas12a. Collectively, these results thus suggest that PAM binding is required for strand separation in dsDNA target and progressive crRNA-TS hybridization, which results in subsequent allosteric activation of Cas12a.

Surface electrostatics of the REC lobe orchestrates NTS cleavage

Previously determined structures of FnCas12a bound to dsDNA targets in pre-cleavage (PDB: 5NFV) and post-cleavage (PDB: 5MGA) states revealed that guide crRNA-TS hybridization displaces 20 nucleotides in the NTS (Stella et al., 2017; Swarts et al., 2017). As the displaced segment of the NTS contains the cleavage site, crRNA-TS hybridization would thus make the NTS susceptible to the ssDNase activity of the RuvC domain. However, there is currently little insight into the mechanism coupling crRNA-TS DNA hybridization to NTS DNA cleavage since an extensive portion of the NTS is structurally disordered in these complexes. To shed light on the mechanism of crRNA-guided target DNA cleavage, we determined an additional structure of a ternary complex comprising wild-type FnCas12a, a 43-nt crRNA, and a target dsDNA comprising full-length target and non-target strands. Although the complex contains the same dsDNA ligand as our previously reported pre-cleavage structure (PDB: 5NFV), the new crystal structure has significantly different unit cell dimensions, indicative of a slightly different conformational state (RMSD: 1.25 Å). The conformation of the structure, determined at 2.65 Å resolution (Table 1), nevertheless corresponds to that of the pre-cleavage complex (PDB: 5NFV, RMSD 1.25 Å). The structure contains a near-complete R-loop in which NTS nucleotides 1-13 and 19-20 are ordered (Figures 3A and 3B). NTS nucleotides 7-13 are bound in a positively charged groove on the outer surface of the RuvC domain by electrostatic contacts with Asn1288 and basic residues including Lys895, Arg1014, Lys1066, Lys1069, Lys1281, and Lys1287 (Figures 3C and S4).

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

Figure 3

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(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, FnCas12aK1218A-K1287A-K1288, and FnCas12aR1014A-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.

To test the importance of the NTS-binding groove residues for target cleavage, we substituted selected amino acid residues in the groove with alanine and generated the mutant proteins FnCas12aR1014A-R1016A-K1066A-K1069A and FnCas12aK1281A-K1287A-N1288A. The cis-cleavage efficiency of FnCas12aK1281A-K1287A-N1288A was only slightly perturbed; FnCas12aR1014A-R1016A-K1066A-K1069A in turn showed greatly reduced cis-cleavage activity (Figure 3D). This suggests that the NTS-binding groove contributes to energetic stabilization of the displaced NTS in the context of the R-loop, thereby promoting crRNA-TS DNA hybridization. Additionally, the NTS-binding groove provides a positively charged conduit connecting the PAM interaction domain and the RuvC active site and thus likely positions the NTS for insertion into the RuvC catalytic site (Figure 3C). The positively charged residues involved in NTS binding are partially conserved across the Cas12a nuclease family and the positively charged NTS-binding groove is a prominent feature in other Cas12a orthologs (Figure S5).

Non-target DNA strand cleavage precedes target DNA strand cleavage

In our previous study, we provided evidence suggesting that Cas12 family enzymes generate double-strand DNA breaks by sequential cleavage of the two substrate DNA strands (Swarts et al., 2017). Based on the observation that the NTS binding groove in FnCas12a appears to guide the displaced NTS into the RuvC catalytic site (Figure 3C), we hypothesized that the NTS is cleaved before the TS during dsDNA cleavage. To test this, we designed modified target dsDNA substrates in which either the TS or the NTS contained multiple backbone phosphorothioate modifications (Figure 4A). Such modifications typically inhibit cleavage by divalent cation dependent nucleases (Eckstein and Gish, 1989). The phosphorothioate modifications did not affect FnCas12a-crRNA binding to the dsDNA targets since FnCas12aE1006Q-crRNA bound both TS-modified and NTS-modified dsDNA targets as efficiently as unmodified control DNAs (Figure S6). Upon incubation with a catalytically active FnCas12a-crRNA complex, we observed that phosphorothioate modification of the TS did not affect cis-cleavage of the NTS (Figure 4B). This indicates that NTS cleavage can occur independently of TS cleavage. In contrast, cis-cleavage of the TS was almost completely abrogated in the dsDNA substrate in which the NTS was modified (Figure 4B), which suggests that the cleavage of the NTS prerequisite for TS cleavage. To verify this, we tested dsDNA targets in which the phosphorothioate-modified NTS was either truncated, nicked, or contained a single unmodified ‘cleavable’ phosphate (Figure 4C). TS cleavage occurred with all of the additional substrates. Of note, when the modified NTS was nicked, the TS was cleaved as efficiently as in the non-modified dsDNA target. Although we cannot rule out the possibility that the presence of the phophorothioate modifications perturbs TS binding in the RuvC active site, this result strongly suggests that the phosphorothioate modifications in the NTS do not interfere with TS cleavage per se, and implies that the NTS has to be cleaved before TS cleavage can occur.

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

Figure 4

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(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.

One possible explanation for the sequentially ordered NTS-TS cleavage is that the presence of an intact NTS in a dsDNA substrate sterically hinders the binding of the TS in the RuvC catalytic site. Alternatively, NTS cleavage might be required for the unwinding of the PAM-distal dsDNA segment, which has to occur before the TS can enter the catalytic site. To investigate if PAM-distal DNA unwinding affects TS cleavage, we tested if partially unwound dsDNA targets could be cleaved regardless of NTS modifications. FnCas12a cleaved the TS in the unwound dsDNA target, albeit at lower efficiencies than for the unmodified dsDNA target (Figure 4D). Based on this result, we conclude that NTS cleavage enables local unwinding of the PAM-distal DNA by fraying (Andreatta et al., 2006), which makes the TS susceptible for cleavage. Together, these observations imply that cis-cleavage of dsDNA targets occurs in an ordered sequential manner in which the displaced NTS is cleaved first and the TS second.

Cas12a releases the PAM-distal end of cleaved target dsDNA

Studies of Cas9 previously revealed that dissociation of cleaved dsDNA targets from the Cas9-guide RNA complex is very slow (Richardson et al., 2016; Sternberg et al., 2014). This is likely a consequence of extensive interactions with both the PAM-proximal and PAM-distal ends of the cleaved dsDNA target. Unlike Cas9, Cas12a cleaves its target DNA distal from the PAM. Consequently, Cas12a engages in very few interactions with the PAM-distal cleavage product of a target dsDNA substrate. To determine the fate of target DNA after Cas12a-mediated cleavage, we utilized dsDNA targets in which one of the four termini (i.e. either the 5’-TS, 3’-TS, 5’-NTS, or 3’-NTS) was labeled with a covalently attached fluorophore. All four target DNAs were efficiently bound by the catalytically inactive FnCas12aE1006Q-R1218A-crRNA complex, as judged by fluorescence-detection size exclusion chromatography (Figure S7). Upon incubation with a catalytically active FnCas12a-crRNA complex, the target DNAs were cleaved (Figure 5). The PAM-proximal cleavage product (i.e. 3’-TS and 5’-NTS labeled DNA) eluted together with the FnCas12a-crRNA complex, indicating that the PAM-proximal DNA remains bound to FnCas12a after cleavage. In contrast, PAM-distal DNA (i.e. 5’-TS and 3’-NTS labeled DNA) did not co-elute with the FnCas12a-crRNA complex, indicating that the PAM-distal DNA is released from the FnCas12a-crRNA complex upon cleavage. These findings are in agreement with recent single-molecule biophysical studies (Singh et al., 2018).

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

Figure 5

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(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 FnCas12aE1006Q-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.

We further hypothesized that release of cleaved target DNA is required to clear the RuvC catalytic site and allow subsequent trans-cleavage of non-target DNA. To test this, we tested the trans-nuclease activity of FnCas12a-crRNA complexes incubated with and dsDNA targets containing phosphorothioate modifications that prevent cis-cleavage (Figure 5E) and thus consequently release of the PAM-distal cleavage product. While dsDNA targets carrying phosphorothioate modifications are efficiently bound by FnCas12a (Figure S6), trans-activation of FnCas12a by these targets was impaired compared to (unmodified) control ssDNA and dsDNA targets (Figure 5F); the TS-modified dsDNA target yielded reduced trans-activation while NTS-modified dsDNA target was incapable of trans-activation. These results indicate that cis-cleavage of dsDNA targets is required for dsDNA-induced trans-nuclease activity and imply that dissociation of the PAM-distal end of the dsDNA target is prerequisite for trans-cleavage of non-target DNA.

Discussion

Cas12-family enzymes exhibit two DNase activity modes; besides crRNA-guided cis-cleavage of complementary DNA targets, these nucleases also catalyze trans-cleavage of non-target ssDNA substrates upon binding and allosteric activation by a complementary DNA target (Chen et al., 2018; Li et al., 2018). The target DNA-induced, trans-acting DNAse activity has been exploited to develop sensitive technologies for nucleic acid detection (Chen et al., 2018; Gootenberg et al., 2018). By combining insights into the cis- and trans-acting deoxyribonuclease activities derived from two FnCas12a-crRNA-DNA structures and biochemical experiments described here with insights from three recent complementary studies (Jeon et al., 2018; Singh et al., 2018; Strohkendl et al., 2018), we provide a revised mechanistic model for both cis- and trans-acting deoxyribonuclease activities of Cas12a enzymes (Figure 6).

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

Figure 6

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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.

Target dsDNA binding is initiated by recognition of the PAM sequence. In line with recent single-molecule biophysical studies (Jeon et al., 2018), our biochemical experiments show that the PAM is dispensable for cleavage of pre-unwound dsDNA targets, indicating that PAM recognition is mechanistically required for strand invasion of the crRNA in the target dsDNA and R-loop formation. A conserved lysine residue is inserted into the dsDNA duplex, possibly initiating TS-NTS unwinding (Swarts et al., 2017). PAM binding additionally introduces a kink in the TS, which further contributes to local strand separation and facilitates base pairing of the TS to the seed segment of the crRNA, while the displaced NTS is stabilized by interactions with the PI domain (Stella et al., 2017; Swarts et al., 2017). Progressive target DNA duplex unwinding is concomitant with crRNA-TS hybridization (Jeon et al., 2018; Singh et al., 2018; Strohkendl et al., 2018), which results in catalytic activation of Cas12a upon formation of a complete R-loop. We show that crRNA-TS DNA hybridization, and not PAM binding per se, drives the allosteric activation of Cas12a by displacing the REC2 domain, thereby exposing the RuvC catalytic site. These findings provide a mechanistic rationale for previous studies demonstrating that 3’ truncations of the crRNA resulted in complete loss of Cas12a activity (Kim et al., 2016; Zetsche et al., 2015). NTS-binding groove that guide the displaced NTS toward the RuvC catalytic site.

Overall, the mechanisms underpinning PAM-dependent target DNA recognition and R-loop formation in Cas12a are strikingly similar to those in Cas9 (Anders et al., 2014; Jiang et al., 2015; Sternberg et al., 2015), and likely a result of convergent evolution, given that Cas9 and Cas12a enzymes have distinct evolutionary histories and consequently different molecular architectures (Shmakov et al., 2017). However, there are notable mechanistic differences between Cas9 and Cas12a besides distinct PAM requirements. In contrast to Cas12a, the PAM is essential for dsDNA target cleavage by Cas9 even if the DNA is pre-unwound (Jeon et al., 2018). Additionally, Cas12a has a lower intrinsic tolerance for crRNA-TS mismatches compared to Cas9 and, as a consequence, requires higher complementarity between the crRNA and the TS before it is catalytically activated (Strohkendl et al., 2018). Importantly, the mechanisms for DNA cleavage differ substantially between the two enzymes; while Cas9 utilizes a HNH and a RuvC domain to cleave the TS and NTS, respectively (Gasiunas et al., 2012; Jinek et al., 2012), Cas12a utilizes a single RuvC domain for cis-cleavage of both target DNA strands as well as for trans-cleavage of non-target DNA.

The structure of FnCas12a R-loop complex reported here reveals that the displaced NTS is coordinated and oriented toward the RuvC catalytic site not only by the PI domain residues identified in earlier studies (Stella et al., 2017; Swarts et al., 2017), but additionally by several positively charged amino acid residues in the RuvC domain that form a structurally conserved NTS-binding groove. This not only contributes to R-loop formation but also primes NTS for cleavage in the RuvC active site. As a result, dsDNA cleavage proceeds through an ordered sequential mechanism in which NTS is cleaved first, followed by the NTS, as recently shown by other studies (Jeon et al., 2018; Strohkendl et al., 2018) and verified by our experiments utilizing non-cleavable dsDNA substrates. Initial occupation of the RuvC catalytic site by the NTS might preclude TS cleavage. However, our experiments show that in the absence of TS-NTS base pairing in the PAM-distal part of the R-loop, the TS can be cleaved even if the NTS is uncleavable. This implies that NTS cleavage is required to facilitate DNA strand separation in the PAM-distal duplex, possibly by fraying of the cleaved NTS ends (Andreatta et al., 2006). Based on similarities with crystal structures of the related Cas12b nucleases (Yang et al., 2016), we speculate that the Nuc domain induces a kink in the TS upon binding, which promotes its insertion into the RuvC catalytic site and subsequent cleavage. Currently available crystal structures of Cas12a reveal substantial conformational flexibility of the Nuc domain with respect to the remainder of the NUC lobe, suggesting that the Nuc domain might play an active role in both unwinding and positioning of the TS. The unspecific ssDNase activity of the RuvC catalytic site of Cas12a can result in further trimming of the ends of the cis-cleaved target dsDNA, resulting in heterogeneous cleavage products. Similar observations have also been made for Cas9 (Stephenson et al., 2018), although Cas9 does not possess trans-nuclease activity. In contrast to Cas9, which contains two catalytic sites buried in its scaffold, Cas12a needs to have its single catalytic site solvent exposed to allow association first with the NTS and subsequently with the TS. We hypothesize that trans-cleavage of non-target ssDNA might be a consequence of the evolutionary history of Cas12 nucleases that has been preserved due to the requirement to catalyze both TS and NTS cleavage using a single active site. Corroborating recent studies (Jeon et al., 2018; Singh et al., 2018; Strohkendl et al., 2018), we finally show that upon sequential cleavage of the two DNA strands of a dsDNA target, the PAM-distal cleavage product is released, while the PAM-proximal cleavage product remains bound to Cas12a. This maintains Cas12a in a catalytically competent state, in which its RuvC active site remains exposed to solvent and is poised to catalyze trans-cleavage of non-target ssDNA substrates. However, the exact mechanism by which ssDNA substrates bind in and are cleaved by the RuvC catalytic site remains to be determined.

The insights provided by this study likely apply to other Cas12a orthologs as well as to Cas12b nucleases. Similar to Cas12a, Cas12b demonstrates cis- and trans-acting deoxyribonuclease activities (Chen et al., 2018). Moreover, the RuvC nuclease domain in Cas12b is responsible for cleavage of both the TS and NTS DNA strands in a dsDNA target (Liu et al., 2017; Yang et al., 2016). These findings are important in the context of the biological function of type V CRISPR-Cas systems in genome defense as it demonstrates that they can target both ssDNA and dsDNA invasive nucleic acid elements. Furthermore, following cleavage of an invading target dsDNA, the persistence of trans-acting DNase activity might contribute to the interference mechanism and invader clearance.

Finally, our mechanistic insights have implications for further development and utilization of Cas12a as a genome editing tool. Cas12a orthologs, including FnCas12a, have successfully been used for genome editing in bacteria, yeast, insects, plants, and mammalian cells, including human cell lines (Swarts and Jinek, 2018). As Cas12a does not only cleave target genomic dsDNA in cis, but also non-target ssDNA in trans, it could potentially target transcription or replication intermediates, or ssDNA templates that are used for homology-directed repair (Richardson et al., 2016). Although Cas12a has a low intrinsic tolerance for off-target cleavage (Strohkendl et al., 2018), the significance of non-target DNA trans-cleavage by Cas12a for genome editing awaits further investigation. The NTS-binding groove might be an interesting target for structure-guided engineering of Cas12a to increase its specificity. It has previously been demonstrated that mutating residues involved in NTS coordination can lower mismatch tolerance in both Cas9 and Cas12a (Chen et al., 2017; Gao et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016). Additional mutations of NTS-binding residues could further enhance the specificity of Cas12a variants. Lastly, asymmetric retention and release of DSB ends upon Cas12a-catalyzed cleavage suggests that editing efficiency or DNA repair outcomes might be dependent on the orientation of the bound Cas12a, particularly in the context of multiplexed genome editing. In conclusion, our studies revealed critical insights into the molecular mechanisms of target DNA binding and both cis- and trans-cleavage of DNA substrates by type V CRISPR effector nucleases, providing a framework for their further development for genome editing applications.

STAR⋆Methods

Key Resource Table.

REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
Potassium chloride Roth 6781.2
HEPES Roth HN78.1
Sodium citrate tribasic dihydrate Fluka 71404
Magnesium chloride Fluka 63064-500G
Potassium thiocyanate Roth P753.1
Bis-Tris propane Sigma-Aldrich B6755-250G
Poly(ethylene glycol) 3,350 – Sigma-Aldrich 202444-500G
Ethylene Glycol Fluka 03750
Proteinase K ThermoFisher Scientific EO0491
Ethylenediaminetetraacetic acid Roth 8040.3
DNaseAlert Integrated DNA Technologies 11-04-02-04
Deposited Data
Atomic coordinates and structure factors This study PDB: 6I1K and 6I1L
Raw image data This study http://dx.doi.org/10.17632/dvvxbf5v2p.1
Experimental Models: Organisms/Strains
E. coli Rosetta (DE3) Novagen 70954-3
Oligonucleotides
List of oligonucleotides used in this study This study Table S1
Recombinant DNA
List of plasmids used in this study This study Table S2
M13mp18 Single-stranded DNA New England Biolabs N4040S
M13mp18 RF I DNA New England Biolabs N4018S
Software and Algorithms
XDS (Kabsch, 2010) http://xds.mpimf-heidelberg.mpg.de/
PHASER (Mccoy et al., 2007) http://www.phaser.cimr.cam.ac.uk/index.php/Phaser_Crystallographic_Software
Coot (Emsley and Lohkamp, 2010) https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PHENIX (Afonine et al., 2012; Terwilliger and Terwilliger, 2004; Terwilliger et al., 2013) http://www.phenix-online.org/
PyMOL The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC https://www.pymol.org/
DynDom (Hayward and Lee, 2002) http://fizz.cmp.uea.ac.uk/dyndom/
PDBePISA (Krissinel and Henrick, 2007) http://www.ebi.ac.uk/pdbe/pisa/
Clustal Omega (Larkin et al., 2007) https://www.ebi.ac.uk/Tools/msa/clustalo/
ESPript 3.0 (Robert and Gouet, 2014) http://espript.ibcp.fr
GraphPad Prism 6 GraphPad Prism 5 for Windows, Version 6.07 www.graphpad.com
ImageQuant TL ImageQuant TL 1D version 8.1 www.gelifesciences.com
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Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Martin Jinek (jinek@bioc.uzh.ch)

Experimental Model and Subject Details

Escherichia coli Rosetta (DE3)

For protein expression, E. coli Rosetta (DE3) was grown in LB at 37°C in a shaker incubator at 120 rpm until an OD600 nm of 0.6 was reached after the temperature was switched to 20°C. After 30 min, expression was induced by addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 0.2 mM. Expression took place at 20°C for 16 h.

Method Details

FnCas12a expression and purification

FnCas12a mutant expression vectors were generated by Gibson assembly using pDS015 as backbone vector, which was amplified using oligonucleotides oDS366 and oDS367 or oDS368 and oDS369. Inserts were generated by annealing two oligonucleotides (oDS364 and oDS365) or ordered as dsDNA gBlock (gbDS02). FnCas12a and FnCas12a mutants were expressed in E. coli Rosetta DE3 and purified as described previously (Swarts et al., 2017). A detailed step-by-step protocol can be found online at bio-protocol (https://bio-protocol.org/e2842).

Crystallization and structure determination

The ternary FnCas12a-crRNA-TS complex was reconstituted by combining purified apo-FnCas12aE1006Q in SEC buffer (500 mM KCl, 20 mM HEPES-KOH (pH 7.5), 1 mM DTT) at a concentration of 10 mg.ml-1 with synthetic crRNA1 (obtained from Integrated DNA Technologies, Table S1) in a 1:1.4 molar ratio (FnCas12a:crRNA1) in the presence of 5 mM MgCl2, and incubating for 10 min at RT. Next, the TS DNA oDS311 (Table S1) was added in a 1:1.4:1.6 ratio (FnCas12a:crRNA1:oDS311). The complex was crystallized at 20°C using the hanging drop vapor diffusion method by mixing equal volumes of protein and reservoir solution. Initial spherulites were obtained at a protein concentration of 8.3 mg.ml-1 with 1.6 M trisodium citrate as reservoir solution. Crystal growth was optimized by iterative microseeding. Data was collected from a crystal that was grown for four weeks at 20°C in a protein concentration of 8.3 mg.ml-1 and 1.6 M trisodium citrate as reservoir solution. Crystals were transferred to a cryoprotectant solution (1.44 M trisodium citrate, 10% (v/v) ethylene glycol) and flash-cooled in liquid nitrogen.

The ternary FnCas12a-crRNA-DNA complex was reconstituted and crystallized as described previously for the FnCas12a-crRNAX-DNA ternary complex (Swarts et al., 2017). Diffraction data was obtained using a crystal grown for four months at 20°C in refinement screens with a final protein concentration of 7.7 mg.ml-1 and a reservoir solution containing 0.1 M Bis-Tris Propane (BTP; pH 6.5), 0.2 M KSCN, and 17.5 % (w/v) polyethylene glycol 3,400. Crystals were transferred to a cryoprotectant solution (0.1 M BTP (pH 6.5), 0.2 M KSCN, 20% (w/v) polyethylene glycol 3,400, 15% (v/v) ethylene glycol, and 5 mM MgCl2) and flash-cooled in liquid nitrogen.

X-ray diffraction data were measured at beamline X06DA (PXIII) of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland). Data were indexed, integrated, and scaled using XDS. Crystals of the FnCas12a-crRNA-TS ternary complex diffracted to a resolution of 2.98 Å and belonged to space group P212121, with two copies of the complex in the asymmetric unit. The structure of the FnCas12a-crRNA-TS complex was solved by molecular replacement in phenix.phaser using a modified FnCas12a-crRNAX-dsDNA (PDB: 5NFV) as search model. Phases obtained using the initial molecular replacement solution were improved by density modification using phenix.resolve (Terwilliger and Terwilliger, 2004) and phenix.morph_model (Terwilliger et al., 2013). The atomic model was built manually in Coot (Emsley and Lohkamp, 2010) and refined using phenix.refine (Afonine et al., 2012). The final binary complex model contains crRNA1 residues (−18)–(+21), target strand residues (-20)-(-1) and FnCas12aE1006Q residues 1–1299, except for residues 550-553, 1009-1013, 1156-1162, and 1277-1279, which lack ordered electron density.

The crystal of the FnCas12a-crRNA-dsDNA ternary complex diffracted to a resolution of 2.65 Å and belonged to space group P21, with one copy per asymmetric unit. The structure was solved by molecular replacement in phenix.phaser using a modified FnCas12a-crRNAX-dsDNA (PDB: 5NFV) as search model. The atomic model was built manually in Coot and refined using phenix.refine. The final ternary complex model contains crRNA residues (−18)–20, target DNA strand nucleotides (−27)–10, and non-target strand nucleotides (−8*)-13* and 19*-29*, and FnCas12a residues 1–1300, except for residues 850-851, 1135-1136, and 1153-1166, which lack ordered electron density.

Structure analysis

Root Means Square Deviations (RMSD) of structure alignments were calculated using Coot LSQ superpose in which main chain atoms were superposed (Emsley and Lohkamp, 2010). Domain movements were analysed using DynDom (Hayward and Lee, 2002). Intramolecular interactions were analysed using PDBePISA (Krissinel and Henrick, 2007). Vector maps were generated using the PyMol script Modevectors.

M13 DNA trans-cleavage experiments

To generate dsDNA targets, TS and NTS oligonucleotides (100 μM, oDS329-oDS342, Table S1) were mixed in a 1:4 molar ratio (TS:NTS). The strands were annealed by incubation at 95°C for 5 min, followed by slow cooling to room temperature. The samples were further diluted in H2O to a concentration of 10 μM. For trans-cleavage experiments, FnCas12a and FnCas12E1006Q (10 μM in SEC buffer) were mixed with crRNA1 (10 μM in H2O) in the presence of 5 mM MgCl2 and incubated at 37°C for 10 min to allow binary complex formation. Target ssDNAs oDS302 or oDS311 (Table S1, 10 μM in H2O) or target dsDNAs (10 μM in H2O) and M13 circular ssDNA or dsDNA substrates (Table S2, 100 ng.μl-1, New England Biolabs) were added. The final reaction (20 μl) contained final concentrations of 0.5x SEC buffer, 2.5 μM FnCas12a or FnCas12aE1006Q, 1.5 μM crRNA1, 5 mM MgCl2, 1.5 μM trans-activating ssDNA or dsDNA, and 10 ng.μl-1 circular DNA substrate. Samples were incubated for 90 min at 37°C. Reactions were stopped by adding EDTA and Proteinase K (Thermo Fisher Scientific) to final concentrations of 80 mM and 0.8 mg.ml-1, respectively, and incubating for 30 min at 37°C. Trans-activating dsDNA was removed from the M13 circular ssDNA using the NucleoSpin® Gel and PCR Clean-up kit (MACHERY-NAGEL) according to the instructions of the manufacturer, with 7-fold excess of NTI DNA binding buffer. 6x DNA loading dye (Thermo Fisher Scientific) was added to the sample and the DNA was resolved on 0.8% (w/v) agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotum) and visualized using a ChemiDoc Touch gel imager (Bio-Rad).

FQ-reporter trans-cleavage assays

To generate dsDNA targets, TS and NTS oligonucleotides (100 μM in H2O; Table S1) were mixed (Table S2) in a 1:49 ratio (final target concentration: 2 μM). The strands were annealed by incubation at 95°C for 5 min, followed by slow cooling to room temperature. For trans-cleavage activity assays, FnCas12a was mixed with crRNA1 (10 μM in H2O) or crRNA-λ (10 μM in H2O) in the presence of 10 mM MgCl2 and incubated at 37°C for 10 min to allow binary complex formation. The sample was diluted 10-fold in dilution buffer (0.5x SEC, 10 mM MgCl2) and 2.5 μl target DNA was added to 50 μl of the protein sample. This sample was mixed with 47.5 μl DNaseAlert™ (IDT) solution (diluted in H2O) in a 384-well plate. The final reaction (100 μl) contained final concentrations of 0.25x SEC buffer, 5 mM MgCl2, 150 nM FnCas12a, 50 nM crRNA, 50 nM DNA trans-activating DNA target and 50 nM DNaseAlert substrate. Fluorescence signal (excitation at 536 nm, emission at 556 nm) was measured over time using a PHERAstar FSX (BMG Labtech) microplate reader with a FP 540-590-590 optical module. Experiments were performed in triplicates. Graphs (displaying averages and standard deviations for triplicates) were generated using GraphPad Prism 6.

Cis-cleavage experiments

The dsDNA targets with modified backbones used in the cis-cleavage experiments shown in Figure 4F were generated by mixing TS and NTS oligonucleotides (100 μM, Table S1) in a 1:2 ratio (unlabeled:Cy5-labeled). The strands were annealed by incubation at 95°C for 5 min, followed by slow cooling to room temperature. The samples were mixed in a 1:1 ratio with native loading dye (0.5x SEC buffer, 5 mM MgCl2, 50% (v/v) glycerol) and resolved on a 10% native polyacrylamide gel. Gel regions containing fluorophore-labeled dsDNA were identified using a Typhoon FLA 9500 gel imager (GE Healthcare) and cut out. The gel was crushed and soaked in H2O for two days before the supernatant was extracted. The dsDNA was isolated from the supernatant by ethanol precipitation and dissolved in H2O. The final concentration of the dsDNA targets was ~0.3 μM.

For all other cis-cleavage experiments, dsDNA targets were generated by mixing Cy5-labeled TS and NTS oligonucleotides (100 μM, Table S1) in a 1:4 ratio (TS-NTS). The strands were annealed by incubation at 95°C for 5 min, followed by slow cooling to room temperature. Samples were diluted to a final dsDNA concentration of 1 μM. FnCas12a or FnCas12aE1006Q-R1218A (10 μM in SEC buffer), crRNA-λ (10 μM), and MgCl2 (50 mM) were mixed and incubated for 10 min at 37°C before the target dsDNA (0.3-1 μM) was added. The final 20 μl reaction contained final concentrations of 0.25x SEC buffer, 2.5 μM FnCas12a or FnCas12aE1006Q-R1218A, 1 μM crRNA-λ, 2.5 mM MgCl2, and 30-100 nM target dsDNA, and was incubated for 30 min at 20°C unless otherwise indicated. Reactions were stopped by adding EDTA and Proteinase K (Thermo Fisher Scientific) to final concentrations of 80 mM and 0.8 mg.ml-1, respectively, and incubating for 30 min at 37°C. Samples were mixed 1:1 with 2x loading dye (5% glycerol (v/v), 90% formamide (v/v), and 2.5 mM EDTA), heated for 10 min at 95°C, and resolved on 20% denaturing (7 M Urea) polyacrylamide gels. Fluorescence of the Cy5-labeled DNA was detected using a Typhoon FLA 9500 gel imager (GE Healthcare). ImageQuant TL 1D (version 8.1) was used to analyse the fluorescence signal strength. Experiments were performed in duplicates. Graphs (displaying averages and standard deviations for duplicates) were generated using GraphPad Prism 6.

High-performance liquid chromatography experiments

Labeled and unlabeled target strand oligonucleotides (100 μM, Table S1) and labeled and unlabeled non-target strand oligonucleotides (100 μM, Table S1) were mixed in a 1:2 ratio (labeled:unlabeled) and annealed by incubation at 95°C for 5 min, followed by slow cooling to room temperature. The formed dsDNA targets were diluted in water to 10 μM. Prior to high-performance liquid chromatography (HPLC) analysis, FnCas12a or FnCas12aE1006Q-R1218A, crRNA-λ (10 μM), MgCl2 (50 mM), SEC buffer, and target DNA (10 μM) were mixed. The resulting 100 μl reaction contained final concentrations of 0.5x SEC buffer, 2 μM FnCas12a or FnCas12aE1006Q-R1218A, 1 μM crRNA-λ, 2.5 mM MgCl2, and 0.25 μM target dsDNA, and was incubated for 1 h at 37°C. Samples were resolved by HPLC on a Superdex 200 5/150 size exclusion column (GE Life Science) with a 3-ml bed volume and 20 bar pressure limit, which was equilibrated and run in 0.5x SEC at a flow rate of 0.1 ml.min-1. Absorption at 280nm and 260nm and the fluorescent signal of Cy5- or ATTO532-labeled DNA were recorded throughout the run. For each target dsDNA, fractions were collected and subsequently analysed by SDS-PAGE for protein content and by 20% denaturing (7 M Urea) PAGE for DNA content. Fluorescence of the Cy5- or ATTO532-labeled DNA was detected using a Typhoon FLA 9500 gel imager (GE Healthcare).

Data and Software Availability

The atomic coordinates and structure factors reported in this paper are deposited in the Protein Data Bank (PDB). The accession numbers for the structures reported in this paper are PDB: 6I1L (FnCas12-crRNA-TS-only complex) and 6I1K (FnCas12-crRNA-TS-NTS complex). The unprocessed image files, image file analysis, and raw microplate reader data used to prepare the figures in this manuscript are deposited in Mendeley Data and are available at http://dx.doi.org/10.17632/dvvxbf5v2p.1.

Supplementary Material

Supplemental Figures and Tables

Acknowledgements

We are grateful to Meitian Wang, Vincent Olieric, and Takashi Tomizaki at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) for assistance with X-ray diffraction measurements. We thank members of the Jinek group for discussions and critical reading of the manuscript. This work was supported by Swiss National Science Foundation (SNSF) Project Grants to M.J. (SNSF 31003A_149393 and 31003A_182567) and by long-term postdoctoral fellowships from the European Molecular Biology Organization (EMBO) to D.C.S (ALTF 179-2015 and aALTF 509-2017). M.J. is International Research Scholar of the Howard Hughes Medical Institute and Vallee Scholar of the Bert L & N Kuggie Vallee Foundation.

Footnotes

Author contributions

D.C.S. and M.J. designed experiments. D.C.S. prepared and crystallized the Cas12a complexes, collected X-ray data, determined crystal structures and carried out biochemical assays. D.C.S. and M.J. wrote the manuscript.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figures and Tables
EMS84915-supplement-Supplemental_Figures_and_Tables.pdf (1.7MB, pdf)

Data Availability Statement

The atomic coordinates and structure factors reported in this paper are deposited in the Protein Data Bank (PDB). The accession numbers for the structures reported in this paper are PDB: 6I1L (FnCas12-crRNA-TS-only complex) and 6I1K (FnCas12-crRNA-TS-NTS complex). The unprocessed image files, image file analysis, and raw microplate reader data used to prepare the figures in this manuscript are deposited in Mendeley Data and are available at http://dx.doi.org/10.17632/dvvxbf5v2p.1.

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