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Idt Crispr Guide Rna Design Tool

Abstract

CRISPR–Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes—in class 1 CRISPR–Cas systems—or domains of a single protein—in class 2 systems1,2,3. Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR–Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii (DiCas7-11), when expressed in Escherichia coli, has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors—and unique among class 1 systems—DiCas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity4,5. This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity.

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Data availability

Sequencing data are available from the Sequence Read Archive under BioProject accession number PRJNA657647.

Code availability

Supporting information and computational tools are available on the Abudayyeh–Gootenberg laboratory website (https://www.abugootlab.org/).

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Acknowledgements

We thank M. Terns, E. Sontheimer and M. Mittens for discussions; A. Badran for discussions and plate-reader assistance; P. Reginato, D. Weston and E. Boyden for MiSeq instrumentation; S. Tonegawa and D. King for centrifuge assistance; G. Feng and D. Wang for gel imager support; G. Paradis and M. Griffin for flow cytometry assistance; A. Sejr Hansen for providing mES cells; and R. Desimone, J. Crittenden and S. Lall for support and discussions. O.O.A. and J.S.G. are supported by NIH grant 1R21-AI149694; The McGovern Institute Neurotechnology (MINT) program; and the McGovern Institute. K.S.M. and E.V.K. are supported by the Intramural Research Program of the National Institutes of Health (National Library of Medicine). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors jointly supervised this work: Omar O. Abudayyeh, Jonathan S. Gootenberg

Affiliations

  1. McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA

    Ahsen Özcan, Rohan Krajeski, Eleonora Ioannidi, Brennan Lee, Apolonia Gardner, Omar O. Abudayyeh & Jonathan S. Gootenberg

  2. ETH Zurich, Zurich, Switzerland

    Eleonora Ioannidi

  3. Harvard University, Harvard Medical School, Virology Program, Boston, MA, USA

    Apolonia Gardner

  4. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

    Kira S. Makarova & Eugene V. Koonin

Contributions

O.O.A. and J.S.G. conceived the study; O.O.A. and J.S.G designed and participated in all experiments. A.Ö. participated in small-RNA-sequencing analyses, protein purifications, and in vitro cleavage and bacterial assays. E.I. and A.G. assisted with mammalian Cas7-11 assays; R.K. helped with cloning and plasmid sequencing; B.L. helped with genome and metagenome mining of new orthologues; K.S.M. and E.V.K. contributed to sequence and phylogeny analyses; O.O.A., E.V.K., J.S.G and A.Ö. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Omar O. Abudayyeh or Jonathan S. Gootenberg.

Ethics declarations

Competing interests

O.O.A. and J.S.G. are co-inventors on a patent application (US 63/073,898) filed by MIT relating to work in this manuscript. O.O.A. and J.S.G. are co-founders of Sherlock Biosciences, Proof Diagnostics, Moment Biosciences and Tome Biosciences. O.O.A. and J.S.G. were advisors for Beam Therapeutics during the performance of the described research.

Additional information

Peer review information Nature thanks Malcolm White and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 Additional type III-E family members and novel type III-D2 loci architecture and multiple alignment of representative orthologues.

a, Loci corresponding to listed accession IDs (see Supplementary Table 1), with key genes highlighted. CRISPR array symbols are not representative of the number of spacers in the array. b, Multiple alignment of representative Cas7-11 orthologues showing conservation of the residues involved in catalysis: D177, D429, D654, D745, D758, and E959. For the D177 alignment, members of the Csm3 family, which are Cas7-like proteins, are included for analysis of conserved residues (highlighted in grey).

Extended Data Fig. 2 Heterologous expression of the Desulfonema ishimotonii Type III-E CRISPR–Cas system and associated CRISPR array.

a, Heterologously expressed Desulfonema ishimotonii Type III-E full locus matures crRNAs. b, Heterologously expressed single effector protein DiCas7-11 matures crRNAs in E. coli. c, Expression of the DiCas7-11 CRISPR array without any effector or accessory proteins. d, Schematic of the DR secondary structure. Scissors denote the cleavage site for processing and maturation. e, Processing of crRNAs occurs when the CJcCas7-11 locus is heterologously expressed in E. coli. Mature 37 nt crRNAs are generated, containing a 15nt DR.

Extended Data Fig. 3 The RNA-guided RNA-targeting Cas7-11 is capable of defence against ssRNA MS2 phage and RNA knockdown in bacteria.

a, Schematic of CRISPR array screen of all spacers targeting the MS2 genome. b, Results of the MS2 interference screen shown as box plots. Enrichment of DiCas7-11 spacers in the phage targeting condition denote survival of bacteria and enhanced representation of specific active spacers. Boxes denotes 25th and 75th percentiles with the median marked by the middle line. The whiskers are calculated via the Tukey method (1.5 times the inter-quartile range). Outliers are denoted by blue plus symbols. c, Results of the MS2 interference screen showing enrichment of DiCas7-11a non-targeting spacers across varying phage dilution amounts as box plots. Boxes denotes 25th and 75th percentiles with the median marked by the middle line. The whiskers are calculated via the Tukey method (1.5 times the inter-quartile range). Outliers are denoted by blue plus symbols. d, Number of DiCas7-11a spacers that display survival enrichment over a threshold of 1.7 across different phage dilution conditions. e, Quantification of resistance conferred by top MS2-targeting DiCas7-11 spacers compared against a panel of 4 non-targeting spacers. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques in the dilution assay. f, Quantification of resistance conferred by two top MS2-targeting DiCas7-11 spacers compared against a panel of 6 non-targeting spacers. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques in the dilution assay. Data are mean ± s.e.m.; n = 3.

Extended Data Fig. 4 Protospacer flanking site (PFS) sequences for DiCas7-11 and effect of accessory proteins on interference.

a, Gating strategy used for all bacterial RFP knockdown experiments in Fig. 1e, g and Extended Data Fig. 6l. b, Schematic for targeting of DiCas7-11 DNA target in a plasmid with resulting sequence motifs. c, Schematic for targeting of DiCas7-11 RNA target in β-lactamase (ampicillin resistance gene) and the resulting PFS determined by 20 depleted targets. d, PFS analysis of top spacers from the MS2 phage screen (e-5 condition) in the 8 bp flanking the target region to the left. e, PFS analysis of top spacers from the MS2 phage screen (e-5 condition) in the 8 bp flanking the target region to the right. f, Schematic of the Type III-E locus of the Desulfonema ishimotonii. g, Phage plaque assay of the Type III-E DiCas7-11 effector alone and as part of the entire locus. Two top MS2 targeting guides along with four non-targeting guides are used to assess target interference and survival against phage. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques in the dilution assay. h, Phage plaque assay of E. coli transformed with the DiCas7-11 locus containing corresponding accessory gene knockouts. A top MS2 targeting guide along with a non-targeting guide is used. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques in the dilution assay. Data are mean ± s.e.m.; n = 3. i, Phage plaque assay of E. coli transformed with either the complete DiCas7-11 locus or DiCas7-11 supplemented with corresponding accessory gene knock-ins. A top MS2 targeting guide along with a non-targeting guide is used to assess target interference and survival against phage. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques in the dilution assay. Data are mean ± s.e.m.; n = 3.

Extended Data Fig. 5 Additional characterization of DiCas7-11 cleavage activity.

a, Purified DiCas7-11 product showing purity at different concentrations. b,DiCas7-11a cleavage of synthetic 31nt MS2 ssRNA with a 31nt crRNA completely duplexed to the target showing cleavage fragments that are generated in the targeting condition with protein (indicated by red triangles). c,DiCas7-11 incubated with a crRNA targeting a single-stranded DNA (ssDNA) target with 5′ labelling of the ssDNA strand. d,DiCas7-11a incubated with a crRNA targeting a double-stranded (dsDNA) target with 5′ labelling of the top strand (left) or bottom strand (right). e,DiCas7-11 incubated with either ssRNA or dsRNA targeting and crRNAs tiling the MS2 target. f, In vitro cleavage of MS2 ssRNA at 37 °C with varying concentrations of DiCas7-11 incubated with a crRNA against the target with and without Csx29 protein. MS2 ssRNA target is 5′ labelled with Cy5. Cleavage bands are marked by asterisks. g, Protospacer flanking site (PFS) sequence screen showing in vitro cleavage of randomized PFS targets and lack of sequence preference flanking the target site for DiCas7-11 cleavage. h, Incubation of a long body-labelled MS2 ssRNA target with DiCas7-11 and targeting or non-targeting crRNA in the presence of different ions or chelating agents, demonstrating dependence on magnesium, manganese, or calcium. i, In vitro cleavage of ssRNA 1 target containing a region of complementarity against the mature DiCas7-11 DR. The crRNA and DiCas7-11 are incubated with targets containing different length regions of complementarity to the DR. The ssRNA target is body labelled with Cy5. j, Cleavage of ssRNA 1 target with increasing amounts of DiCas7-11-crRNA complex from 0 nM to 233 nM. The ssRNA target is body labelled with Cy5. k, Cleavage of ssRNA 1 target at increasing incubation periods from 0 min to 180 min. The ssRNA target is body labelled with Cy5.

Extended Data Fig. 6 Additional biochemical characterization of Cas7-11.

a, Schematic showing sequence of DiCas7-11 crRNA 1, targeting the ssRNA 1 target. b, Cleavage of ssRNA 1 target with crRNA 1 of varying DR and spacer lengths. c, Schematic showing sequence of DiCas7-11 crRNA 1, targeting the long MS2 target. d, Cleavage of long MS2 target with crRNA 1 with mutagenized DRs. e,DiCas7-11:crRNA complex binding to a complementary MS2 ssRNA target is determined by electrophoretic mobility shift assay (EMSA). EMSAs are performed for wild-type (WT) DiCas7-11, dead DiCas7-11, a non-targeting crRNA, and a MS2-targeting guide alone. f,DiCas7-11:crRNA complex binding to a complementary MS2 ssDNA target is determined by EMSA. g, Quantification of band intensities for non-targeting guide EMSA in e showing fitted curve (mean ± s.e.m.; n = 3). h, Quantification of band intensities for active DiCas7-11 with MS2 targeting guide EMSA in e showing fitted curve (mean ± s.e.m.; n = 3). i, Quantification of band intensities for MS2 targeting guide EMSA gel in e showing fitted curve (mean ± s.e.m.; n = 3). j, Quantification of band intensities for dead DiCas7-11 EMSA in e showing fitted curve (mean ± s.e.m.; n = 3). k, Quantification of band intensities for ssDNA EMSA gel in f showing fitted curve (mean ± s.e.m.; n = 3). l, RFP knockdown in E. coli is assayed via flow cytometry with wild-type and D429A/D654A DiCas7-11 protein expression. Data are mean ± s.e.m.; n = 3. m, Quantification of resistance conferred by top MS2-targeting DiCas7-11 spacers with active DiCas7-11, dead DiCas7-11, and no protein. Resistance is quantified as the highest surviving titer of MS2 phage that generates plaques (mean ± s.e.m.; n =3 ).

Extended Data Fig. 7 Additional characterization of DiCas7-11 processing.

a, CRISPR array processing assay showing robust processing activity by DiCas7-11 at concentrations ranging from 0 to 233 nM. b, Schematic of in vitro transcription of pre-crRNA and processing by DiCas7-11. c, Sequence of single spacer pre-crRNA, showing locations of mutated bases. d, Processing of transcribed pre-crRNA, showing the effect of single position mutations on processing. e, Processing of transcribed pre-crRNA at different concentrations of DiCas7-11. f, Processing of pre-crRNA by DiCas7-11 in the presence of different ions or chelating agents. CRISPR array processing by DiCas7-11 is ion independent. g, Processing activity of a synthetic DiCas7-11 CRISPR array by DiCas7-11 protein with predicted catalytic processing mutants in the protein insert region with a characteristic KxYxH catalytic triad motif.

Extended Data Fig. 8 Further characterization of RNA knockdown in mammalian cells by Cas7-11.

a, Knockdown of Gluc mRNA in mammalian cells by DiCas7-11 with a panel of guides tiled across the transcript. b, Knockdown of Gluc mRNA in mammalian cells by DiCas7-11 with N- and C-terminus fusions of nuclear export signal (NES) tags (Adeno type 5 E1B-55K) with guides tiled across the transcript. c, Knockdown activity in HEK293FT cells of DiCas7-11, shRNA, LwaCas13a, PspCas13b, and RfxCas13d against the Gluc transcript normalized to corresponding non-targeting controls. All guides are designed to target the same region of the Gluc transcript. d, Comparison of knockdown activity of Gluc mRNA in mammalian cells between active DiCas7-11, catalytically inactive D429A/D654A DiCas7-11, and GFP. Guides have the full DR sequence. e, Knockdown activity of DiCas7-11 against the multiple endogenous transcripts normalized to two non-targeting conditions. Dotted lines represent background expression and 65% knockdown by DiCas7-11. f, Knockdown of EGFP and mCherry mRNA in mammalian cells by DiCas7-11 with a panel of tiling guides. g, Knockdown of Gluc mRNA in mammalian cells by DiCas7-11 with a panel of guides of different lengths. h, Knockdown of Gluc mRNA in mammalian cells by hvsCas7-11 with a panel of guides containing the full DR sequence. Guides are designed to be tiled across the Gluc transcript. i, Knockdown of Gluc mRNA in HepG2 cells with hvsCas7-11 and no protein conditions using guides 1 and 11 as well as a non-targeting guide. j, Knockdown of Gluc mRNA in HEK293FT cells with hvsCas7-11 modified with an NES sequence (Adeno type 5 E1B-55K) and using Gluc guide 1. The NES tag is either C-terminally fused or fused on both ends of the hvsCas7-11 protein. All data in this figure are mean ± s.e.m.; n = 2 or n = 3, as shown.

Extended Data Fig. 9 Development of d DiCas7-11 for programmable RNA-editing in mammalian cells and further characterization of specificity.

a, RNA editing via d DiCas7-11-NES-ADAR2 fusions programmed to target a specific adenosine via a cytidine mismatch. b,DiCas7-11 guide design for programmable A-to-I editing. The mismatch distance is defined as the position of the A-C mismatch relative to the 3′ end of the guide. c, RNA A-to-I editing of Cypridina luciferase (Cluc) mRNA W85X mutation in mammalian cells by dDiCas7-11-NES-ADAR2. Guides have mismatch distances between 2–50nt. Editing is measured either by restoration of Cluc luciferase activity normalized to the non-targeting guide condition (top) or percentage conversion of the adenosine to inosine (bottom). d, RNA A-to-I editing of Cluc mRNA W85X mutation in mammalian cells with different nuclear localization tags and linker architectures compared to ADAR deaminase without DiCas7-11 fusion. Data are mean ± s.e.m.; n = 3. e, End point readout of collateral activity of either DiCas7-11 or LwaCas13a targeting and non-targeting crRNA against MS2 ssRNA target. f, RFP knockdown by DiCas7-11 or LwaCas13a in E. coli. Corresponding growth curves are shown in Fig. 4b. g-h, Effects on cell health by Gluc targeting with CRISPR effectors in (g) HEK293FT cells and (h) U87 glioblastoma cells. i, Number of significant off-targets when targeting the Gluc transcript in HEK293FT and U87 cells by DiCas7-11, shRNA, LwaCas13a, PspCas13b, and RfxCas13d using transcriptome-wide RNA-sequencing data. Off-targets are determined by significance testing of differentially expressed transcripts between targeting and non-targeting guide conditions. j, Number of significant off-targets when targeting the Gluc transcript in HEK293FT and U87 cells by DiCas7-11, shRNA, LwaCas13a, PspCas13b, and RfxCas13d using transcriptome-wide RNA-sequencing data. Off-targets are determined by significance testing of differentially expressed transcripts between the targeting guide condition and cells only expressing EGFP. Data are mean ± s.e.m.; n = 3.

Extended Data Fig. 10 Model of Cas7-11 expression, processing, and interference.

a, Schematic of Cas7-11 expression, processing, and interference against ssRNA viruses and other ssRNA targets. b, Comparison of Cas7-11 features to characteristics of Cas13 and type III-A/B systems. c, Hypothetical scenario for the evolution of a single-protein CRISPR–Cas effector from a multi-subunit effector complex. Cas7-11, a single-effector programmable RNase combining the pre-crRNA processing and target-cleaving RNase activities, apparently originated from two type III-D variants. Three Cas7 domains (domains 3, 4 and 5) are derived from subtype III-D2 that contains a Cas7x3 effector protein along with Cas10 and another Cas7-like domain fused to a Cas5-like domain. The origin of the N-terminal Cas7 and putative Cas11 domain of Cas7-11 is not entirely clear, but most likely, these domains are derived from a III-D1 variant, where both genes are stand-alone3.

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Özcan, A., Krajeski, R., Ioannidi, E. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature 597, 720–725 (2021). https://doi.org/10.1038/s41586-021-03886-5

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Idt Crispr Guide Rna Design Tool

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