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Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Abstract

Although CRISPR-Cas9 nucleases are widely used for genome editing1,2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3,4,5,6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis7. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

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Figure 1: Evolution and characterization of SpCas9 variants with altered PAM specificities.
Figure 2: SpCas9 PAM variants robustly modify endogenous sites in zebrafish embryos and human cells.
Figure 3: A D1135E mutation improves the PAM recognition and spacer specificity of SpCas9.
Figure 4: Characterization of St1Cas9 and SaCas9 in bacteria and human cells.

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Sequence Read Archive

Data deposits

All new reagents described in this work have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas). A web-tool to design sgRNA sites for the engineered variants and orthogonal Cas9 nucleases described in this study can be found at http://www.CasBLASTR.org. The sequences generated in this study have been deposited in the Sequences Read Archive under accession number SRP058629.

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Acknowledgements

We thank D. Edgell for providing the bacterial strain and plasmids related to the bacterial selection; J. Angstman and V. Pattanayak for discussion and comments on the manuscript. This work was supported by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378) and NIH R01 GM107427 to J.K.J., NIH R01 GM088040 to J.K.J. and R.T.P., The Jim and Ann Orr Research Scholar Award (to J.K.J.), and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to B.P.K.).

Author information

Authors and Affiliations

Authors

Contributions

B.P.K., M.S.P., S.Q.T. and N.T.N. performed all bacterial and human cell-based experiments. A.P.W.G. and Z.L. performed all zebrafish experiments. S.Q.T., V.T., Z.Z. and M.J.A. analysed the site-depletion, targeted deep-sequencing, and GUIDE-seq data. B.P.K., R.T.P., J.-R.J.Y. and J.K.J. directed the research and interpreted experiments. B.P.K. and J.K.J. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to J. Keith Joung.

Ethics declarations

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Extended data figures and tables

Extended Data Figure 1 Bacterial-based positive selection used to engineer altered PAM specificity variants of SpCas9.

a, Expanded schematic of the positive selection from Fig. 1b (left panel), and validation that SpCas9 behaves as expected in the positive selection (right panel). b, Schematic of how the positive selection was adapted to select for SpCas9 variants that have altered PAM recognition specificities. A library of SpCas9 clones with randomized PAM-interacting (PI) domains (residues 1097–1368) is challenged by a selection plasmid that harbours an altered PAM. Variants that survive the selection by cleaving the positive selection plasmid are sequenced to determine the mutations that enable altered PAM specificity.

Extended Data Figure 2 Amino acid sequences of clones that cleave target sites bearing alternate PAMs in the bacterial-based positive selection system.

a, Sequences of variants that survived >10% when re-tested in the positive selection assay against an NGA PAM site (see Methods). Variants were selected from libraries containing randomly mutagenized PAM-interacting domains (residues 1097–1368) with or without a starting R1335Q mutation. Sequence differences compared with wild-type SpCas9 are highlighted. The histogram represents the number of changes at each position (not counting the starting R1335Q mutation). b, Sequences of variants that survived >10% when re-tested in the positive selection assay against a site containing an NGC PAM. Variants were selected from libraries containing randomly mutagenized PAM-interacting domains (residues 1097–1368) with starter mutation pairs of R1335E/T1337R or R1335T/T1337R. Sequence differences compared with wild-type SpCas9 (shown at the top) are highlighted. The histogram below illustrates the number of changes at each position (not counting starter mutations at R1335 or T1337).

Extended Data Figure 3 Bacterial cell-based site-depletion assay for profiling the global PAM specificities of Cas9 nucleases.

a, Expanded schematic illustrating the negative selection from Fig. 1d (left panel), and validation that wild-type SpCas9 behaves as expected in a screen of sites with functional (NGG) and non-functional (NGA) PAMs (right panel). b, Schematic of how the negative selection was used as a site-depletion assay to screen for functional PAMs by constructing negative selection plasmid libraries containing 6 randomized base pairs in place of the PAM. Selection plasmids that contain PAMs cleaved by a Cas9/sgRNA of interest are depleted while PAMs that are not cleaved (or poorly cleaved) are retained. The frequencies of the PAMs following selection are compared to their pre-selection frequencies in the starting libraries to calculate the post-selection PAM depletion value (PPDV). c, d, A cutoff for statistically significant PPDVs was established by plotting the PPDV of PAMs for catalytically inactive SpCas9 (dCas9) (grouped and plotted by their second/third/fourth positions) for the two randomized PAM libraries (c). A threshold of 3.36 standard deviations from the mean PPDV for the two libraries was calculated (red lines in (d)), establishing that any PPDV deviation below 0.85 is statistically significant compared to dCas9 treatment (red dashed line in (c)). The grey dashed line in (c) indicates a fivefold depletion in the assay (PPDV of 0.2).

Extended Data Figure 4 Concordance between the site-depletion assay and EGFP disruption activity.

Data points represent the average EGFP disruption of the two NGAN and NGNG PAM sites for the VQR and EQR variants (Fig. 1g) plotted against the mean PPDV observed for library 1 and 2 (Fig. 1f) for the corresponding PAM. The red dashed line indicates PAMs that are statistically significantly depleted (PPDV of 0.85, see Extended Data Fig. 3c), and the grey dashed line represents fivefold depletion (PPDV of 0.2). Mean values are plotted with the 95% confidence interval.

Extended Data Figure 5 Structural and functional roles of D1135, G1218, and T1337 in PAM recognition by SpCas9.

a, Structural representations of the six residues implicated in PAM recognition. The left panel illustrates the proximity of D1135 to S1136, a residue that makes a water-mediated, minor groove contact to the third base position of the PAM12. The right panel illustrates the proximity of G1218, E1219, and T1337 to R1335, a residue that makes a direct, base-specific major groove contact to the third base position of the PAM12. Angstrom distances indicated by yellow dashed lines; non-target strand guanine bases dG2 and dG3 of the PAM are shown in blue; other DNA bases shown in orange; water molecules shown in red; images generated using PyMOL from PDB:4UN3. b, Mutational analysis of six residues in SpCas9 that are implicated in PAM recognition. Clones containing one of three types of mutations at each position were tested for EGFP disruption with two sgRNAs targeted to sites harbouring NGG PAMs. For each position, we created an alanine substitution and two non-conservative mutations. S1136 and R1335 were previously reported to mediate contacts to the third guanine of the PAM12, and D1135, G1218, E1219, and T1337 are reported in this study. EGFP disruption activities were quantified by flow cytometry; background control represented by the dashed red line; error bars represent s.e.m., n = 3.

Extended Data Figure 6 Insertion or deletion mutations induced by the VQR SpCas9 variant at endogenous zebrafish sites containing NGAG PAMs.

For each target locus, the wild-type sequence is shown at the top with the protospacer highlighted in yellow (highlighted in green if present on the complementary strand) and the PAM is marked as red underlined text. Deletions are shown as red dashes highlighted in grey and insertions as lower case letters highlighted in blue. The net change in length caused by each indel mutation is shown on the right (+, insertion; –, deletion). Note that some alterations have both insertions and deletions of sequence and in these instances the alterations are enumerated in parentheses. The number of times each mutant allele was recovered (if more than once) is shown in brackets.

Extended Data Figure 7 Endogenous human genes targeted by wild-type and evolved variants of SpCas9.

a, Sequences targeted by wild-type, VQR, and VRER SpCas9 are shown in blue, red, and green, respectively. Sequences of sgRNAs and primers used to amplify these loci for T7E1 are provided in Supplementary Tables 1 and 2. b, Mean mutagenesis frequencies detected by T7E1 for wild-type SpCas9 at eight target sites bearing NGG PAMs in the four different endogenous human genes (corresponding to the annotations in panel a). Error bars represent s.e.m., n = 3.

Extended Data Figure 8 Specificity profiles of the VQR and VRER SpCas9 variants determined using GUIDE-seq7.

The intended on-target site is marked with a black square, and mismatched positions within off-target sites are highlighted. a, The specificity of the VQR variant was assessed in human cells by targeting endogenous sites containing NGA PAMs: EMX1 site 4, FANCF site 1, FANCF site 3, FANCF site 4, RUNX1 site 1, RUNX1 site 3, VEGFA site 1, and ZNF629. b, The specificity of the VRER variant was assessed in human cells by targeting endogenous sites containing NGCG PAMs: FANCF site 3, FANCF site 4, RUNX1 site 1, VEGFA site 1, and VEGFA site 2.

Extended Data Figure 9 Activity differences between D1135E and wild-type SpCas9.

a, Mutagenesis frequencies detected by T7E1 for wild-type and D1135E SpCas9 at six endogenous sites in human cells. Error bars represent s.e.m., n = 3; mean fold change in activity is shown. b, Titration of the amount of wild-type or D1135E SpCas9-encoding plasmid transfected for EGFP disruption experiments in human cells. The amount of sgRNA plasmid used for all of these experiments was fixed at 250 ng. Two sgRNAs targeting different EGFP sites were used; error bars represent s.e.m., n = 3. c, Targeted deep-sequencing of on- and off-target sites for 3 sgRNAs using wild-type and D1135E SpCas9. The on-target site is shown at the top, with off-target sites listed below highlighting mismatches to the on-target. Fold decreases in activity with D1135E relative to wild-type SpCas9 at off-target sites greater than the change in activity at the on-target site are highlighted in green; control indel levels for each amplicon are reported. d, Mean frequency of GUIDE-seq oligo tag integration at the on-target sites, estimated by restriction fragment length polymorphism analysis. Error bars represent s.e.m., n = 4. e, Mean mutagenesis frequencies at the on-target sites detected by T7E1 for GUIDE-seq experiments. Error bars represent s.e.m., n = 4. f, GUIDE-seq read count comparison between wild-type SpCas9 and D1135E at 3 endogenous human cell sites. The on-target site is shown at the top and off-target sites are listed below with mismatches highlighted. In the table, a ratio of off-target activity to on-target activity is compared between wild-type and D1135E to calculate the normalized fold changes in specificity (with gains in specificity highlighted in green). For sites without detectable GUIDE-seq reads, a value of 1 has been assigned to calculate an estimated change in specificity (indicated in orange). Off-target sites analysed by deep-sequencing in panel c are numbered to the left of the EMX1 site 3 and VEGFA site 3 off-target sites.

Extended Data Figure 10 Additional PAMs for St1Cas9 and SaCas9 and activities based on spacer lengths in human cells.

a, PPDV scatterplots for St1Cas9 comparing the sgRNA complementarity lengths of 20 and 21 nucleotides obtained with a randomized PAM library for spacers 1 and 2 (see also Fig. 4a). PAMs were grouped and plotted by their third/fourth/fifth/sixth positions. The red dashed line indicates PAMs that are statistically significantly depleted (see Extended Data Fig. 3c) and the grey dashed line represents fivefold depletion (PPDV of 0.2). b, Table of PAMs with PPDVs of less than 0.2 for St1Cas9 under each of the four conditions tested. PAM numbering shown on the left is the same as in Fig. 4a. c, PPDV scatterplots for SaCas9 comparing the sgRNA complementarity lengths of 21 and 23 nucleotides obtained with a randomized PAM library for spacers 1 and 2 (see also Fig. 4b). PAMs were grouped and plotted by their third/fourth/fifth/sixth positions. The red and grey dashed lines are the same as in a. d, Table of PAMs with PPDVs of less than 0.2 for SaCas9 under each of the four conditions tested. PAM numbering shown on the left is the same as in Fig. 4b. e, Human cell EGFP disruption activities of St1Cas9 and SaCas9 at sites of various spacer lengths. Frequencies were quantified by flow cytometry; error bars represent s.e.m., n = 3 or 4; mean level of background EGFP loss represented by the dashed red line. f, Activity for all replicates of data shown in e plotted against spacer length. n = 3 or 4; bars illustrate mean and 95% confidence interval; number of sites per spacer length indicated. g, Activity for all replicates shown in Fig. 4d, e, plotted against spacer length. n = 3 or 4; bars illustrate mean and 95% confidence interval; number of sites per spacer length indicated.

Supplementary information

Supplementary Information

This file contains Supplementary Sequences and details of the Supplementary software for analyzing PAM depletion MiSeq data. (PDF 830 kb)

Supplementary Table 1

This table shows the Oligos used to generate positive and negative selection plasmids. (XLSX 14 kb)

Supplementary Table 2

This file contains S. pyogenes sgRNAs targets. (XLSX 20 kb)

Supplementary Table 3

This file contains the PAM MiSeq data. (XLSX 2014 kb)

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Kleinstiver, B., Prew, M., Tsai, S. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015). https://doi.org/10.1038/nature14592

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