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Trans-nuclease activity of Cas9 activated by DNA or RNA target binding

Nature Biotechnology volume 43, pages 558–568 (2025) Cite this article

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Abstract

Type V and type VI CRISPR–Cas systems have been shown to cleave nonspecific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) in trans, but this has not been observed in type II CRISPR–Cas systems using single guide RNA. We show here that the type II CRISPR–Cas9 systems directed by CRISPR RNA and trans-activating CRISPR RNA dual RNAs show RuvC domain-dependent trans-cleavage activity for both ssDNA and ssRNA substrates. Cas9 possesses sequence preferences for trans-cleavage substrates, preferring to cleave T- or C-rich ssDNA substrates. We find that the trans-cleavage activity of Cas9 can be activated by target ssDNA, double-stranded DNA and ssRNA. The crystal structure of Cas9 in complex with guide RNA and target RNA provides a structural basis for the binding of target RNA to activate Cas9. Based on the trans-cleavage activity of Cas9 and nucleic acid amplification technology, we develop the nucleic acid detection platforms DNA-activated Cas9 detection and RNA-activated Cas9 detection, which are capable of detecting DNA and RNA samples with high sensitivity and specificity.

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Fig. 1: Cas9 displays trans-cleavage activity to poly(T)/poly(C) ssDNA homopolymers.
The alternative text for this image may have been generated using AI.
Fig. 2: crRNA- and tracrRNA-guided trans-cleavage activity of Cas9.
The alternative text for this image may have been generated using AI.
Fig. 3: Analysis of nonspecific ssDNA sequence and structural preference.
The alternative text for this image may have been generated using AI.
Fig. 4: Target binding activates the trans-cleavage activity of SpyCas9.
The alternative text for this image may have been generated using AI.
Fig. 5: Specificity and sensitivity of Cas9-mediated nucleic acid detection.
The alternative text for this image may have been generated using AI.

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Data availability

The atomic coordinates included in this study have been deposited in the PDB with the following accession codes: 8KAG ref. 50, 8KAH, 8KAI, 8KAJ, 8KAK, 8KAL and 8KAM. PDB IDs 4OO8 and 5U33 were used for Fig. 1a and Extended Data Fig. 9a. PDB ID 7Z4J was used for Extended Data Fig. 8a,d. PDB ID 4ZT0 was used for Extended Data Fig. 9a,b. Source data are provided with this paper.

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Acknowledgements

We are grateful to the staff of the BL-18U1, BL-19U1, BL-10U2 and BL-02U1 beamlines at the National Center for Protein Sciences Shanghai at Shanghai Synchrotron Radiation Facility. We thank W. Han for providing the plasmids and C. Wu, Y. Wu, J. Hu and W. Zheng for technical support. This work was supported by grants from the National Natural Science Foundation of China (32022047), the Natural Science Foundation of Fujian Province of China (2023J01023 and 2023J05008) and the Science and Technology Project of Xiamen City (3502Z20227020).

Author information

Author notes

  1. These authors contributed equally: Jiyun Chen, Ying Chen, Linglong Huang.

Authors and Affiliations

  1. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, China

    Jiyun Chen, Ying Chen, Linglong Huang, Xiaofeng Lin, Hong Chen, Wenwen Xiang & Liang Liu

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  1. Jiyun Chen
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Contributions

J.C., Y.C. and L.H. expressed and purified the proteins and grew crystals. H.C., X.L., Y.C., W.X. and L.L. collected X-ray diffraction data. L.L. solved the structures. J.C., Y.C., L.H., X.L. and H.C. performed all of the cloning and the biochemical assays. J.C. and L.L. prepared the figures. L.L. wrote the manuscript and supervised all of the research.

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Correspondence to Liang Liu.

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Nature Biotechnology thanks Piyush Jain and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Cas9 shows similar trans-cleavage activity to Cas12a.

a-c, Denaturing gel showing the trans-cleavage of Cy3-labeled homopolymeric ssDNAs (poly A, poly T, poly C and poly G) by FnCas12a (a), FnCas9 (b), and SauCas9 (c) in the presence of a target ssDNA activator. All assays in Extended Data Fig. 1 were repeated three times independently with similar results.

Source data

Extended Data Fig. 2 Kinetic analysis of crRNA-tracrRNA and sgRNA guided trans-cleavage.

a, Representative ssDNA-FQ standard curve for kinetic parameters determination. b-e, Plots of fluorescence intensity versus time for crRNA-tracrRNA (b) or sgRNA (d) guided trans-cleavage, using target ssDNA01 activator and increasing ssDNA-FQ substrate concentrations. Michaelis-Menten fits for crRNA-tracrRNA (c) or sgRNA (e) guided ssDNA-FQ trans-cleavage using target ssDNA01 activator. The concentrations of SpyCas9 used in assays were listed in graph. f-g, Comparison of the kinetic parameters of crRNA-tracrRNA and sgRNA guided trans-cleavage with ss/dsDNA02 (f) or ss/dsDNA03 (g) activator. kcat/Km values are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test.

Source data

Extended Data Fig. 3 Trans-cleavage of non-specific ssDNA directed by crRNA and tracrRNA.

a, Comparison of sgRNA and crRNA-tracrRNA guided FnCas9 trans-cleavage on M13 phage ssDNA substrate using a target ssDNA activator. There is no sequence homology between the M13 ssDNA and the target ssDNA. b, crRNA-tracrRNA guided SpyCas9 trans-cleavage assays using 5′-Cy5 labeled non-specific dsDNA-1 substrate with random sequence. c, crRNA-tracrRNA guided SpyCas9 trans-cleavage assays using 5′-Cy5 labeled non-specific ssDNA-2 (left) and 3′-Cy5 labeled non-specific ssDNA-3 (right) substrates with random sequences. Assays in Extended Data Fig. 3a–c were repeated three times independently with similar results. d, Schematic representation of an assay to explore the effect of the trans-cleavage activity of Cas9 on bacterial immunity. e, SpyCas9 can target and delete the pTarget plasmid in Escherichia coli. The pCas9 and pTarget plasmids contain the Chl-resistant gene and Amp-resistant gene, respectively. The cells were serially diluted and dropped onto plates containing indicated antibiotics. Control: The guide sequence of crRNA is not complementary to the target sequence (pTarget). Targeting: The guide sequence of crRNA is complementary to the target sequence (pTarget). f, The trans-cleavage activity of SpyCas9 does not prevent plague formation. Ten-fold serial dilutions of M13 phage were dropped onto bacterial lawns. g, The titers of M13 phage after infecting Escherichia coli. PFU values are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test.

Source data

Extended Data Fig. 4 Effects of substrate DNA sequence and secondary structure on trans-cleavage activity.

a, Schematic of long non-specific ssDNA substrates. The DNA sequences of LT2, LT3 and LT4 are derived from the genomes of Microviridae sp. isolate SD_SC_96 (Genbank: MH572378.1), Microviridae sp. isolate ctzV77 (Genbank: BK042979.1), and Microvirus mar36 (Genbank: MZ089782.1), respectively. The single-stranded poly T regions of substrates are colored blue. Poly T to poly A mutations are induced in LA2, LA3 and LA4 corresponding to LT2, LT3 and LT4, respectively. Poly A regions of substrates are colored green. Poly T to poly TC mutation is induced in LTC4 corresponding to LT4. Poly T to poly TA mutation is induced in LTA4 corresponding to LT4. b-c, Testing the crRNA-tracrRNA induced trans-cleavage activity of SpyCas9 (b) or FnCas9 (c) on long non-specific ssDNA substrates containing poly T, poly A, poly TC or poly TA sequences. All data in Extended Data Fig. 4b,c are shown as mean ± s.d. (n = 3 independent experiments).

Source data

Extended Data Fig. 5 The RuvC domain is responsible for the trans-cleavage activity of SpyCas9.

a-c, Denaturing gel showing target dsDNA cis-cleavage (a), target ssDNA cis-cleavage (b), and non-specific ssDNA trans-cleavage (c) by SpyCas9 wild-type (WT), RuvC-dead mutant (D10A) or HNH-dead mutant (H840A). dsDNA and ssDNA substrates were labeled with Cy5 or Cy3. Assays in Extended Data Fig. 5 were repeated three times independently with similar results.

Source data

Extended Data Fig. 6 Substrate sequence preference of Cas9 cis-cleavage.

a, Denaturing gel showing the cleavage of target strand (TS) of target dsDNA. The target strands of target dsDNAs were all labeled with 6-FAM. Poly T, poly A, poly G and poly C in the target strands are highlighted in green, magenta, red and limon, respectively. Cleavage site of target strand is indicated by blue triangle. b, Denaturing gel showing the cleavage of non-target strand (NTS) of target dsDNA. The non-target strands of target dsDNAs were all labeled with 6-FAM. Poly T, poly A, poly G and poly C in the non-target strands are highlighted in green, magenta, red and limon, respectively. Cleavage site of non-target strand is indicated by blue triangle. c, (Top) Sequences of target dsDNA plasmids and complementary guide RNAs used in reactions. Poly T and poly G in the non-target strands are highlighted in green and magenta, respectively. Cleavage site of non-target strand is indicated by blue triangle. (Bottom) Agarose gel analysis showing the DNA cleavage by SpyCas9 with indicated target dsDNA. Assays in Extended Data Fig. 6 were repeated three times independently with similar results.

Source data

Extended Data Fig. 7 The formation of an 18-nt guide-target DNA duplex is essential for the complete activation of Cas9.

a-c, (Top) Fluorescence time-course assays showing the sensitivity of SpyCas9-catalyzed trans-cleavage using 13-nt (a), 11-nt (b), and 8-nt (c) ssDNA-FQ substrates and increasing target activator concentrations. (Bottom) Comparison of slopes of curves within 10-25 min from the top time-course graphs. The slope was calculated by simple linear regression. Data are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test. d, (Top) Comparison of trans-cleavage rates within 10-20 min for SpyCas9 using dsDNA activators with indicated mismatches. (Bottom) Sequences of dsDNA activators and the complementary guide RNAs used in reactions. Data are shown as mean ± s.d. (n = 3 independent experiments). e-f, Time-course assays (e) and maximum fluorescence signals (f) showing that the DNA target length affects the trans-cleavage activity of SpyCas9. Data are shown as mean ± s.d. (n = 3 independent experiments).

Source data

Extended Data Fig. 8 Structural differences between SpyCas9-sgRNA-NTS_TS and SpyCas9-crRNA-tracrRNA-NTS_TS complexes.

a, Structures of SpyCas9-sgRNA-NTS_TS complexes with the 16-nt (top), 17-nt (middle first) and 18-nt (middle second) target strands in same orientation. (Bottom) The structure of SpyCas9-sgRNA-NTS_TS18 complex in catalytic state (PDB: 7Z4J). b, Structures of SpyCas9-crRNA-tracrRNA-NTS_TS complexes with the 16-nt (upper), 17-nt (middle) and 18-nt (bottom) target strands. c, SEC purification of SpyCas9-sgRNA-NTS_TS18 and SpyCas9-crRNA-tracrRNA-NTS_TS18 complexes. SEC assays were repeated three times independently with similar results. d, Structural comparison of the REC1 and HNH domains, as well as the sgRNA and target DNA in the checkpoint and catalytic state complexes. The checkpoint complex structure is shown in gray. In addition to the magenta core of the REC1 domain, the catalytic complex structure is colored according to a.

Source data

Extended Data Fig. 9 Comparison of structures of SpyCas9-sgRNA-target ssDNA and SpyCas9-sgRNA-target ssRNA.

a, Structural comparison between the SpyCas9-sgRNA-target ssDNA (PDB: 4OO8) complex and the SpyCas9-sgRNA (PDB: 4ZT0) complex. Vector length correlates with the domain motion scale. b, Structural comparison between the SpyCas9-sgRNA-target ssRNA complex and the SpyCas9-sgRNA complex (PDB: 4ZT0). c, Superposition of the SpyCas9-sgRNA-target ssRNA complex onto the SpyCas9-sgRNA-target ssDNA complex. The structure of ssDNA bound state is colored in silver. Protein domains of SpyCas9-sgRNA-target ssRNA are colored according to Fig. 4g. d, Structural overlay of the little variation domains in ssRNA bound state and ssDNA bound state complexes. e, Structural overlay of the domains that underwent marked conformational change in ssRNA bound state and ssDNA bound state complexes. f, Structural overlay of nucleic acids in ssRNA bound state and ssDNA bound state complexes. g, Superposition of the guide RNA (orange)-target ssRNA (megenta) duplex onto an A-form RNA duplex (gray).

Extended Data Fig. 10 Applications of Cas9-detection.

a-b, Fluorescence time-course assays showing the sensitivity of RACD (a) and DACD (b) to mpox (CB) strain. c, The net fluorescence intensity showing the specificity of DACD to mpox (CB) strain. Data are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test. d, Comparison of trans-cleavage activity of SpyCas9 using dsDNA activators with indicated mismatches (Top). Sequences of the dsDNA activators and the complementary guide RNAs used in reactions (bottom). Data are shown as mean ± s.d. (n = 3 independent experiments). e-f, Fluorescence time-course assays showing the sensitivity of RACD (e) and DACD (f) to EGFR T790M mutant. g, Sensitivity analysis of DACD for detecting the EGFR T790M mutant minor allele in mock cell-free DNA samples. Data are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test. h, Rader charts compare the features related to the application of trans-cleavage activity of Cas9 (left), Cas12a (middle) and Cas13a (right) in nucleic acid detection. Each of the 7 features form individual axes varying from 0 (without the feature) to 100 (with the feature). The vertexes of the smaller gray heptagon indicate 50 (feature can be realized by artificial modification). The value of each feature is depicted by a small circle, except that the 0 point is not displayed. The area bounded by connecting the data values for each axis is filled with the corresponding color (Cas9: purple, Cas12a: green, Cas13a: blue).

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Chen, J., Chen, Y., Huang, L. et al. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat Biotechnol 43, 558–568 (2025). https://doi.org/10.1038/s41587-024-02255-7

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