A compact Cascade–Cas3 system for targeted genome engineering

Abstract

CRISPR–Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7–424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade–Cas3, but not Cas9. A transferable ‘all-in-one’ vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR–Cas use was enhanced with an ‘anti-anti-CRISPR’ strategy. P. aeruginosa Type I-C Cascade–Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

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Fig. 1: Type I-C CRISPR-mediated self-targeting leads to genomic deletions.
Fig. 2: Optimization and characterization of Cascade–Cas3-directed genomic editing.
Fig. 3: Iterative generation of multiple genomic deletions in P. aeruginosa.
Fig. 4: Cascade–Cas3-mediated heterologous editing in various bacteria.
Fig. 5: Cascade–Cas3-mediated gene editing in native settings.

Data availability

Plasmids pJW31 and pCas3cRh are available through Addgene (numbers 136423 and 133773, respectively). Raw WGS data associated with Figs. 1d, 3b, 4a,c,f and 5a) have been uploaded to GenBank (accession numbers CP047061CP047079) and are also available, along with bacterial strains, upon request from the corresponding author. P. aeruginosa strains available for laboratories with BSL-2 clearance. Source data are provided with this paper.

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Acknowledgements

B.C. is supported by the Eötvös National Scholarship of Hungary and a Marie Skłodowska-Curie Actions Individual Global Fellowship (‘GenDels’, no. 844093) of the Horizon 2020 Research Program of the European Commission. L.M.L. is supported by the HHMI Gilliam Fellowship for Advanced Study and the UCSF Discovery Fellowship. Research on plant immunity in the Lewis laboratory is supported by the USDA grant nos. ARS 2030-21000-046-00D and 2030-21000-050-00D (J.D.L.), and the NSF Directorate for Biological Sciences grant no.IOS-1557661 (J.D.L.). I.J.C.-L. is supported by a Grace Kase fellowship from UC Berkeley and the NSF Graduate Research Fellowship Program. A.V.R. is supported by funding from the Pew Charitable Trusts. E.D.C. is funded by the Chan Zuckerberg Biohub. CRISPR–Cas3 projects in the Bondy-Denomy Laboratory are supported by the UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation, the Innovative Genomics Institute and an NIH Director’s Early Independence Award DP5-OD021344. We thank J.B. Goldberg (Emory University) for providing the plasmid pJM230, and A. Borges (UCSF) for providing pAB01 to clone Type I-F crRNAs. We thank the Bondy-Denomy laboratory for productive conversations pertaining to this project.

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Authors

Contributions

B.C. and L.M.L. participated in designing and performing experiments, analyzing data, acquiring funding for the project and writing the manuscript. I.J.C.-L. performed in vitro and in planta P. syringae experiments. A.V.-R. performed and analyzed Type I-F CRISPR–Cas editing experiments. J.D.B. assisted in designing and constructing the all-in-one pCas3cRh vector. C.M. constructed the PAO1IIA strain and Cas9 gRNA expression vector. E.D.C. performed WGS. J.D.L. designed experiments with P. syringae. J.B.-D. conceived the study, designed experiments, analyzed data, acquired funding for the project and wrote the manuscript.

Corresponding author

Correspondence to Joseph Bondy-Denomy.

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Competing interests

J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics and a scientific advisory board member and cofounder of Acrigen Biosciences. J.B.-D., L.M.L. and B.C. have filed a patent application relating to various aspects of Cas3-based genome editing.

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Peer review information Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Type I-C CRISPR targeting leads to genomic deletions.

a, Comparison of Type I-C CRISPR system from P. aeruginosa used in the study, to various other previously identified I-C systems from a range of different bacteria. Values show query coverage and percent identity (ID) percentages comparing the four genes of the P. aeruginosa system to each of the other four. * Denotes the reference Type I-C CRISPR system referred to in Ref. 1. b, PCR amplification of a 3 kb genomic fragment flanking the phzM gene targeted using two different crRNAs, phzM_1 and phzM_2. Colony PCRs were performed on 18 biological replicates of self-targeted strains for each crRNA. The PAO1ICparental strain is used as a positive control (wt). L indicates a 1 kb DNA ladder. Source data

Extended Data Fig. 2 Excision of plasmid-encoded spacer sequences.

a, Phage targeting assays with survivors that had no discernable deletion of the crRNA-targeted genomic site. Strains were transformed with a D3 phage-targeting crRNA to assay for IC CRISPR-Cas3 activity. Three unique survivors were isolated from six self-targeting assays for a total of 18 survivors. Control is a non-targeting crRNA. b, Schematic of spacer excision events where the two direct repeats recombine, resulting the loss of the targeting spacer. c, PCR amplification of the crRNA sequence from plasmids isolated from 17 non-deletion self-targeted survivors (selected from 3 biological replicates of 12 analyzed colonies (see Fig. 2a). Pl indicates the original plasmid as the PCR template, Ni indicates a sample where the crRNA was not induced, L indicates a 1 kb DNA ladder. d, Sample chromatogram of a sequenced plasmid with the spacer flipped out. Only one 32 bp repeat sequence remains in the plasmid, the 34 bp spacer sequence and other 32 bp repeat are missing. Source data

Extended Data Fig. 3 Phage-targeting assays to confirm CRISPR-Cas functionality.

a, Phage-targeting assay showing the activity of the modified repeat crRNA constructs. Ten-fold serial dilutions of DMS3 phage and D3 phage were spotted on lawns of PAO1IC expressing either empty vector (top), a crRNA targeting D3 with WT direct repeats (middle), or a crRNA targeting D3 with modified repeats (bottom). b, Phage targeting assay of five non-deletion self-targeting survivors expressing a D3 phage targeting crRNA. Unsuccessful targeting of phage indicates a non-functional CRISPR-Cas system in these strains. The parental PAO1IC strain with a functional CRISPR-Cas system was used as a control. Source data

Extended Data Fig. 4 Genomic targeting of essential gene rplQ.

a, Growth curves of 36 PAO1IC biological replicates targeting the essential gene, rplQ, using the MR crRNA plasmid. b, Phage targeting assays with eight isolated rplQ-targeted survivors to assay for I-C CRISPR-Cas activity. Serial dilutions of DMS3 phage and D3 phage were spotted on lawns of PAO1IC expressing a crRNA targeting phage D3. The parent PAO1IC strain expressing a D3 targeting crRNA (top left) was used as a positive control, while PAO1IC expressing a non-targeting crRNA was used as a negative control. Source data

Extended Data Fig. 5 Genomic targeting using a Type II-A CRISPR-Cas system.

Growth of self-targeting strains of PAO1IIA expressing a self-targeting gRNA targeting the genome at phzM (Ind.). An empty vector (E.V.) and a non-induced phzM targeting strain (N.I.) were used as controls. Mean OD values measured at 600 nm are shown for 8 biological replicates each, error bars indicate SD values.

Extended Data Fig. 6 Genomic deletions and junction sites.

a, Deletion efficiencies observed over six cycles of iterative self-targeting. Six genomic targets were targeted in six different orders. Six survivors were analyzed using site-specific PCR after each cycle, for a total of 36 analyzed colonies (6*6) after each cycle, error bars represent standard deviations. b, Deletion junctions at XNES6 target site of the 6 PAO1IC strains with 6 iterative targeting events each. Sequences of each specific microhomology for the junctions are shown for each strain above the bars representing the given genomes at both ends, deletion sizes are shown below dashed lines for each strain. c, PCR analysis using a representative set of primers amplifying various large deletion junctions (at XNES1, 6, 8, and 9 regions) of the whole-genome sequenced Δ62 strain. Δ62 served as a positive control template, while wtC represents untargeted PAO1IC cells scraped from a lawn of colonies from a single overnight culture grown on plates serving as templates, and wtG represents isolated genomic DNA from a different 1.5 ml overnight culture of untargeted PAO1IC used as templates. Bands appearing for the XNES9 deletion junction for the PAO1IC samples were aspecific and when sequenced, did not match any genomic region of the PAO1IC genome. L indicates a 1 kb DNA ladder. Source data

Extended Data Fig. 7 Genomic targeting of P. aeruginosa PAO1 with all-in-one vector pCas3ch.

a, Map of the I-C CRISPR-Cas all-in-one plasmid pCas3cRh carrying I-C crRNA and genes cas3, cas5, cas8, and cas7 under the control of the rhamnose-inducible rhaSR-PrhaBAD system. b, Growth curve of PAO1 transformed with the pCas3cRh vector expressing a self-targeting crRNA targeting phzM (Ind.). An empty vector (E.V.) and a non-induced phzM targeting strain (N.I.) were used as controls. Mean OD values measured at 600 nm are shown for six biological replicates each. c, Deletion efficiencies for WT PAO1 using the all-in-one vector pCas3cRh carrying all necessary components of the I-C CRISPR-Cas system. Values are averages of three replicates where 12 individual colonies were analyzed using site-specific PCR. Error bars show standard deviations. d, Transformation efficiencies with self-targeting pCas3cRh vectors expressing crRNAs for phzM or XNES 2 compared to a non-targeting control (green bar) in PAO1. Values are means of 3 replicates each, error bars represent SD values.

Extended Data Fig. 8 Genomic targeting of Pseudomonas syringae and growth phenotypes of deletion strains.

a, Growth of P. syringae DC3000 strains expressing the I-C system and distinct crRNAs. Constructs VI, IV-IX, and VIII target P. syringae DC3000 non-essential chromosomal genes, non-targeting crRNA (NT), empty vector (EV). b, Percentage of survivors with targeted deletions in clusters of non-essential virulence effector genes in P. syringae pv. tomato DC3000. Values are averages of three biological replicates where 12 individual colonies were analyzed using site-specific PCR for each, error bars show standard deviations. c, In vitro growth of cluster VI deletion strains in King’s medium B (KB). ΔCEL is the previously published polymutant, while ΔCVI-1 and ΔCVI-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. d, In vitro growth of cluster IV, cluster IX deletion strains in KB. ΔCEL is the previously published polymutant, while ΔCIVΔCIX-1 and ΔCIVΔCIX-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. e, In vitro growth of cluster X deletion strains in KB. ΔCEL is the previously published polymutant, while ΔCX-1 and ΔCX-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. f, In vitro growth of cluster VI deletion strains in apoplast mimicking minimal media (MM). ΔCEL is the previously published polymutant, while ΔCVI-1 and ΔCVI-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. g, In vitro growth of cluster IV, cluster IX deletion strains in MM. ΔCEL is the previously published polymutant, while ΔCIVΔCIX-1 and ΔCIVΔCIX-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. h, In vitro growth of cluster X deletion strains in MM. ΔCEL is the previously published polymutant, while ΔCX-1 and ΔCX-2 are Cas3-generated mutants. Values shown are the means of 4 biological replicates each, error bars represent standard deviations. Source data

Extended Data Fig. 9 CRISPR-Cas3 editing in Klebsiella pneumoniae.

a, Growth curves of K. pneumoniae strains expressing distinct crRNAs targeting rfaH and sacX (2 each). Non-targeting crRNA expressing control is marked in blue. Values depicted are averages of 8 biological replicates each. b, Representative gel electrophoresis of PCR fragments amplified from 8 total surviving colonies each from the 4 crRNA targeting constructs (representing 1 biological replicate of 3 total). Primer pairs amplified regions flanking the targeted position at rfaH and sacX. Wild-type KPPR1 (wt) colonies were used as controls, L represents 1 kb DNA marker ladder. c, Percentage of survivors with targeted deletions at the targeted genomic positions. Values are averages of three biological replicates where 8 individual colonies were analyzed using site-specific PCR for each, error bars show standard deviations. d, Colony morphologies of deletion candidate strains of rfaH and sacX compared to wild-type K. pneumoniae KPPR1. Source data

Extended Data Fig. 10 Genomic editing in native host of Type I-C CRISPR-Cas system and effect of I-C specific anti-CRISPR protein on the process.

a, Editing efficiencies for the Pseudomonas aeruginosa environmental isolate naturally expressing the Type I-C cas genes, transformed with a plasmid targeting phzM with WT repeats or modified repeats. Each data point represents the fraction of isolates with the deletion out of ten isolates assayed. b, Genotyping results for the Pseudomonas aeruginosa environmental isolate using the 0.17 kb HDR template. 10 biological replicates were assayed. Larger band corresponds to the WT sequence, smaller band corresponds to a genome reduced by 0.17 kb. c, Genotyping results of PAO1IC AcrC1 lysogens after self-targeting induction in the presence or absence of aca1 and a non-targeted control. Ten biological replicates per strain were assayed. gDNA was extracted from each replicate and PCR analysis for the phzM gene (targeted gene, top row of gels) or cas5 gene (non-targeted gene, bottom row) was conducted. Only cells that co-expressed aca1 with the crRNA showed loss of the phzM band, indicating genome editing. All replicates had a cas5 band, indicating successful gDNA extraction and target specificity for the phzM locus. Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables 1–4

Tables listing XNES regions of P. aeruginosa PAO1 genome, genomic coordinates of large deletions generated in the study, summary of HR-mediated deletion experiments using I-F CRISPR–Cas system, and table of all oligonucleotides used in the study.

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Unprocessed gel electrophoresis picture of Extended Data Fig. 9b and unprocessed gel electrophoresis picture of Extended Data Fig. 9b

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Unprocessed gel electrophoresis pictures of Extended Data Fig. 10

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Csörgő, B., León, L.M., Chau-Ly, I.J. et al. A compact Cascade–Cas3 system for targeted genome engineering. Nat Methods 17, 1183–1190 (2020). https://doi.org/10.1038/s41592-020-00980-w

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