Harnessing type I CRISPR–Cas systems for genome engineering in human cells

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Abstract

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5,6,7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8,9,10,11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

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Fig. 1: Engineering a FokI–Cascade fusion complex to generate DNA double-strand breaks.
Fig. 2: Genome editing in human cells via RNP and plasmid-based delivery of FokI–EcoCascade.
Fig. 3: Editing efficiency as a function of FokI linker, interspacer length, and Cascade homolog.
Fig. 4: Mismatches between paired gRNAs and target DNA inhibit genome editing by FokI-PseCascade.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request as well as under the BioProject ID PRJNA573067.

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Acknowledgements

We thank employees of Caribou Biosciences for critical reading of the manuscript. J.v.d.O. is supported by the Netherlands Organization for Scientific Research (NWO) through a TOP grant (714.015.001). S.J.J.B. is supported by European Research Council (ERC) Starting grant, number 639707.

Author information

S.E.K., S.C.S., S.H.S., and B.W.K. generated all RNPs. S.E.K., S.H.S., and P.D.D. performed RNP biochemical cleavage experiments. P.C., S.E.K., S.C.S., S.H.S., and B.V. performed RNP nucleofection experiments. P.C., M.M.C., A.M.L., B.V., and D.B.N., with assistance and advice from E.M.S., performed all plasmid-based editing experiments. M.M.C., L.E.S., and S.G. cloned all plasmids. P.C., M.M.C., and A.M.L. generated all gRNAs used in the plasmid-based experiments. T.R., R.K., C.L., A.L.G.O., and C.K.F. provided all computational support. L.M.B., C.W., M.S.T., and M.J.I. prepared libraries for NGS. M.J.I. performed SITE-Seq assay experiments. M.S.T. performed target-enrichment for Cascade–Cas3 experiments. T.K., J.v.d.O., S.J.B., and A.P.M. performed early proof-of-concept experiments. S.B.K. provided experimental guidance and advice. P.C. and S.H.S. jointly oversaw all experiments, analyzed the data, and wrote the manuscript, with input from the other authors. All authors discussed the data and approved the final manuscript.

Correspondence to Peter Cameron or Samuel H. Sternberg.

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Competing interests

The authors declare conflicts of interest. P.C., M.M.C., S.E.K., A.M.L., S.C.S., B.V., P.D.D., T.R., B.W.K., D.B.N., R.K., L.M.B., C.W., M.S.T., M.J.I., L.S.E., C.L., A.L.G.O., E.M.S., C.K.F., S.G., S.B.K., A.P.M., and S.H.S. are current or former employees of Caribou Biosciences, a CRISPR genome editing company, and such individuals may own stock or stock options in Caribou Biosciences. Patent applications have been filed describing this work. Correspondence and requests for materials should be addressed to P.C. (pscameron@cariboubio.com) and S.H.S. (shsternberg@gmail.com). All other authors have no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Design and purification of FokI-EcoCascade.

a, X-ray crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target (PDB ID: 5H9F)(Nature 530, 499–503 (2016)). The PAM is highlighted in the rotated view on the left, and the FokI domain (grey) is shown fused to the N-terminus of Cas8 via a linker sequence (red). This attachment point positions the nuclease domain in close proximity to dsDNA within the interspacer, such that FokI dimerization can occur between two adjacent, oppositely oriented FokI-Cascade monomers. The dsDNA flanking the PAM has been artistically extended beyond the experimental structure, for clarity. b, SDS-PAGE analysis of six purified FokI-Cas8 fusion proteins containing various linker lengths (left of ladder), and three Cas8-less Cascade subcomplexes containing either the λ1, λ2, or both λ1 and λ2 gRNAs (right of ladder). The complex with paired gRNAs (λ1+λ2) was purified from an E. coli strain in which a single CRISPR array encoded three repeats and both spacer sequences. FokI-Cas8 linker sequences were as follows: 8, TGPGAAAR; 10, GGSGSSGGSG; 12, TGPGAAARAASG; 15, GGSGSSGGSGSSGGS; 16, SGSETPGTSESATPES; 30, SGSETPGTSESATPESGGSGSSGGSGSSGG. aa, amino acid. *, contaminating protein. Experiment was repeated > two independent times with similar results; the uncropped gel can be found in Source Data Fig. 1.

Supplementary Figure 2 Biochemical characterization of dsDNA cleavage by FokI-EcoCascade.

a, Design of the target plasmid, in which λ1 and λ2 target sites flanked interspacer DNA in a “PAM-in” configuration. b, Plasmid DNA cleavage as a function of ionic strength. The target plasmid from a, or a non-target plasmid lacking the λ1 and λ2 target sites, was incubated with dimeric FokI-EcoCascade complexes for 30 minutes at 37 °C in a reaction buffer containing NaCl at either 50, 100, 150, or 200 mM. At low salt concentrations, both non-target and target plasmid DNA substrates are non-specifically nicked and degraded, as evidenced by the appearance of low-molecular weight DNA at the bottom of the gel (Deg, degraded DNA). At higher salt concentrations, the non-target plasmid remains intact in its supercoiled state, whereas the target plasmid is linearized upon DSB formation. FokI-Cas8 in these reactions contained a 30-aa linker. c, Plasmid DNA cleavage as a function of interspacer and FokI-Cas8 linker length. The non-target plasmid or target plasmids with interspacers ranging from 20–50-bp were incubated with FokI-EcoCascade containing FokI-Cas8 linker lengths of 8, 10, 12, 15, 16, or 30 aa. The 30-bp interspacer substrate is primarily linearized, whereas shorter or longer interspacer substrates are efficiently nicked but not converted into linear form. The longest 30-aa linker length is still able to produce linearized DNA for longer interspacer substrates, and was thus selected for subsequent experiments. d, DNA DSBs require the presence of Cas8-less Cascade and FokI-Cas8, both paired gRNAs, and both target half-sites. Control reactions, which contained neither target site or only one half-site, or only one of the two paired gRNAs, failed to yield a linearized DNA product. The target plasmid was efficiently cleaved either by co-incubation with FokI-Cas8 and two separately purified Cas8-less Cascade subcomplexes containing either gRNA (lane 10), or by FokI-Cas8 and a single Cas8-less Cascade subcomplex purified from an expression strain encoding both paired gRNAs (λ1+λ2; lane 11). e, The target plasmid is no longer cleaved when both half-sites contain mutations in either the PAM or the seed sequence. f, Target plasmid cleavage as a function of FokI-EcoCascade concentration, and comparison to Cas9-sgRNA. 150 ng plasmid DNA was incubated with protein-RNA complexes ranging from ~3–100 nM for 30 minutes at 37 °C, prior to agarose gel electrophoresis. g, Target plasmid cleavage as a function of incubation time. 200 ng plasmid DNA was incubated with 200 nM FokI-EcoCascade or Cas9-sgRNA complexes at 37 °C from 0–30 minutes, prior to agarose gel electrophoresis. N, nicked; L, linear; SC, supercoiled. All experiments in this panel were repeated two independent times with similar results; uncropped gels can be found in Source Data Fig. 2.

Supplementary Figure 3 Engineering a FokI-EcoCascade fusion complex to generate DNA double-strand breaks within human genomic targets.

A plasmid substrate (left) was incubated with NdeI and dimeric FokI-EcoCascade complexes targeting one of 16 adjacent sites containing interspacers ranging from 25–45 bp. Agarose gel electrophoresis reveals the expected cleavage products in each case. Interspacer distances for each target are shown at the top. Target 3–7 digestions are identical to those in Fig. 1d. N, nicked; L, linear; SC, supercoiled; P, dropout product. Experiment was repeated two independent times with similar results; uncropped gels can be found in Source Data Fig. 2.

Supplementary Figure 4 DNA repair outcomes of DSBs introduced by FokI-EcoCascade.

a-c, Next-generation sequencing data showing that indels are centered at the expected cut site across three additional genomic targets tested in Fig. 2a. a, Target 3 from Fig. 2a. b, Target 5 from Fig. 2a. c, Target 9 from Fig. 2a. The expected cleavage site and both half-sites targeted by the paired gRNAs are displayed. Ref, reference sequence; insertion lengths are given next to the blue bars.

Supplementary Figure 5 DNA repair outcomes are not influenced by FokI-EcoCascade delivery approach.

Heat map showing DNA repair outcomes at three target sites in HEK293 cells. FokI-EcoCascade was delivered as either a pre-assembled ribonucleoprotein complex (RNP), 6-plasmid mixture (6-P), or 2-plasmid mixture (2-P). Deletions lengths from 1–50-bp are in blue, and insertion lengths from 1–8 bp are in green; single bp insertions are separated by nucleotide identity, and the color intensity scales with frequency. The bar graph above the heat map displays total editing efficiency. Rep denotes replicates from independent nucleofections; heat maps were generated from data reported in Fig. 2a,d.

Supplementary Figure 6 Testing “PAM-out” target configurations by fusing FokI to Cas6.

EcoCascade-FokI editing efficiency as a function of FokI-Cas6 linker length and interspacer distance after nucleofection with a paired gRNA expression plasmid and a polycistronic plasmid encoding all 5 proteins separate by 2A peptides. For FokI-Cas6 samples, each data point represents the average of three independent nucleofections at a unique “PAM-out” target site in HEK293 cells, n=4 unique target sites per interspacer distance. Positive control nucleofections with FokI-EcoCascade containing a 30-aa FokI-Cas8 linker and targeting “PAM-in” site 7 from Fig. 2a is shown in grey. No editing was observed for any FokI-Cas6 fusion constructs with the “PAM-out” configuration.

Supplementary Figure 7 FokI-EcoCascade editing at an extended panel of genomic target sites.

FokI-EcoCascade editing efficiencies at 49 sites in HEK293 cells after nucleofection with a paired gRNA expression plasmid and a polycistronic plasmid encoding all 5 proteins separate by 2A peptides, n=3 nucleofection replicates per site, bar graph and error bars report the mean and s.d. across nucleofections. All targets contained 30-bp interspacer distances and AAG or ATG PAM sequences.

Supplementary Figure 8 High-throughput PCR generation of paired gRNA templates for genome editing.

a, Oligo-templated PCR strategy to generate amplicons for paired gRNA expression from a human U6 (hU6) promoter in mammalian cells. The Rev inner oligonucleotide encodes both gRNA sequences and is modified for new target sites, whereas the remaining primers are invariant. b, Editing efficiencies at Target 7 (from Fig. 2) after co-nucleofecting HEK293 cells with the polycistronic plasmid encoding FokI-EcoCascade and either a paired gRNA expression plasmid or paired gRNA expression amplicon, n=1 nucleofection.

Supplementary Figure 9 Expanded screening of FokI-Cas8 linker length and interspacer distance for three Cascade homolog variants.

Editing efficiency as a function of FokI-Cas8 linker length and interspacer distance for FokI-PseCascade (a), FokI-EcoCascade (b), and FokI-SthCascade (c). Each dot represents a single target site in HEK293 cells, n=6 (23-bp), 7 (24-, 30-, 32-33-bp), 8 (25-29-, 31-, 34-bp) in a, b and n=7 (27-, 34-bp), 8 (23-26-, 28-33-bp) in c, bar graph reports the mean across sites. All FokI-PseCascade and FokI-EcoCascade targets contained AAG PAMs, all FokI-SthCascade targets contained GAA PAMs, data in a with 17-aa FokI-Cas8 linker length are identical to those shown in Fig. 3c.

Supplementary Figure 10 Expanded screening of PAM specificity for three Cascade homolog variants.

a, FokI-PseCascade (with 17-aa FokI-Cas8 linker) editing efficiency as a function of PAM sequence. Target sites contained a fixed ATG PAM and a variable PAM at the second half-site, as shown on the x-axis. Each dot represents a single target site in HEK293 cells and n=6 (ATG), 8 (AAC, GAG, ATA, AAT), 9 (AGG), 12 (AAG), 14 (AAA) unique target sites, bar graph and error bars report mean and s.d. across sites, data with dual-AAG/ATG PAMs were identical to those shown in Fig. 3d. b, FokI-EcoCascade (with 17-aa FokI-Cas8 linker) editing as a function of PAM sequence. Target sites contained a fixed AAG PAM and a variable PAM at the second half-site, as shown on the x-axis. Each dot represents a single target site in HEK293 cells and n=6 (AAG), 8, (ATA), 9 (GAG, AAC), 10 (CGC), 11 (CCG), 12 (ATG), 13 (AGG), 15 (AAA) unique target sites, bar graph and error bars report mean and s.d. across sites. c, Same as in b, but with one PAM fixed as an ATG, n=6 (ATG), 8 (AAC, GAG, AAT, ATA), 9 (AGG), 12 (AAG), 14 (AAA) unique target sites, bar graph and error bars report mean and s.d. across sites, data with dual-AAG/ATG PAMs were identical to those shown in b. d, FokI-SthCascade (with 17-aa FokI-Cas8 linker) editing as a function of PAM sequence. Target sites contained a fixed GAA PAM and a variable PAM at the second half-site, as shown on the x-axis. Each dot represents a single target site in HEK293 cells and n=18 (TA), 28 (CC), 30 (AA), 31 (GA), 33 (CA) unique target sites, bar graph and error bars report mean and s.d. across sites.

Supplementary Figure 11 FokI-PseCascade reconstitution across multiple cell types.

Heat map showing editing efficiencies at two target sites (targets 69 and 73 from Supplementary Table 8) in HEK293, U2OS, and HeLa cells. FokI-PseCascade with 17-aa FokI-Cas8 linker was co-expressed with a paired gRNA expression plasmid. Deletions lengths from 1–50-bp are in blue, and insertion lengths from 1–8 bp are in green; single bp insertions are separated by nucleotide identity, and the color intensity scales with frequency. The bar graph above the heat map displays total editing efficiency. Rep denotes replicates from independent nucleofections.

Supplementary Figure 12 Comparison of editing efficiency and DNA repair outcomes with FokI-PseCascade, FokI-dCas9, and wild-type Cas9.

Heat map (left) showing representative DNA repair outcomes at a set of target sites at the AAVS1 and CD34 loci with FokI–PseCascade, FokI-dCas9, and Cas9. FokI-PseCascade targets contained 30-32 bp interspacers and AAG PAMs, FokI-dCas9 targets contained 16 bp interspacers and NGG PAMs, and Cas9 was directed to the right half-site of each FokI-dCas9 target. Targets are shown on the x-axis, and sites with < 5% total indels were excluded from this analysis. Deletions lengths from 1–50-bp are in blue, and insertion lengths from 1–8 bp are in green; single bp insertions are separated by nucleotide identity, and the color intensity scales with frequency. The bar graph above the heat map displays total editing efficiency. Editing efficiency for all targets tested is shown at the top right, each dot represents the average of three nucleofection replicates at a single target site (except FokI-dCas9 target 4, which had two nucleofections), n=15 (FokI-dCas9, Cas9), 16 (FokI-PseCascade) unique target sites, bar graph and error bars report the mean and s.d. across sites.

Supplementary Figure 13 Cell viability and editing specificity associated with the FokI domain.

Cell viability and editing as a function of plasmid nucleofection in HEK293 cells for SpyCas9, PseCascade, FokI-PseCascade, or FokI-PseCas8 after 2 (a) or 5 (b) days of expression. Cytotoxicity (left) is shown on the y-axis, as measured with the CellTiter-Glo® Luminescent Cell Viability Assay, quantifying ATP as an indication of cell viability; editing efficiency (right) was measured with NGS. SpyCas9 was co-expressed with a gRNA expression plasmid (target AAVS1 from Supplementary Tables 18-19); FokI-PseCascade, PseCascade, and FokI-PseCas8 were co-expressed with a paired gRNA expression plasmid (Target 73 from Supplementary Table 8, n=3 nucleofection replicates per concentration except FokI-PseCas8 day 2 viability measurements, where n=1). c, Editing efficiency at several targets (x-axis) in HEK293 cells after nucleofecting with either FokI-dCas9 or FokI-PseCascade and gRNAs directed to either the left half-site and an irrelevant half-site (left), the right half-site and an irrelevant half-site (right), or both half-sites (left+right); FokI-dCas9 targets were from a previous study (Nat Biotechnol 32, 569–576 (2014)), n=3 nucleofection replicates per target site, bar graph and error bars report mean and s.d. across nucleofections.

Supplementary Figure 14 Expression and heterologous reconstitution of PseCascade-PseCas3 in HEK293 cells to generate long-range deletions.

a, Location of eight PseCascade-PseCas3 gRNA target sites in TRAC exon 1. b, HEK293 cells were nucleofected with PseCascade-PseCas3 followed by NGS analysis of edited sites after 4 days. The number of unique deletion classes, relative to an untransfected reference control, were calculated for the eight target sites (x-axis) shown in a, n=2 nucleofection replicates per target site. c, HEK293 cells were nucleofected with PseCascade-PseCas3 targeting site 2 in a followed by analysis of long-range deletions (black) after 5 days, n=2 nucleofection replicates; deletions from one representative replicate are shown. Deletions were enriched using oligo bait probes spanning ~ 2kb surrounding the target site. Deletions < 4 bp were filtered from this display.

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Cameron, P., Coons, M.M., Klompe, S.E. et al. Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nat Biotechnol 37, 1471–1477 (2019) doi:10.1038/s41587-019-0310-0

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