Continuous evolution of SpCas9 variants compatible with non-G PAMs


The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer–sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.

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Fig. 1: PANCE of SpCas9 binding activity on non-G PAMs.
Fig. 2: Selection improvement enables evolution of SpCas9 variants with robust activity on non-G PAMs.
Fig. 3: Comprehensive characterization of PAM preferences using a genomically integrated human cell target sequence library.
Fig. 4: Mammalian cell indel formation and DNA specificity of evolved Cas9 variants.
Fig. 5: Mammalian cytosine and adenine base editing activity and scope of evolved variants and SpCas9-NG.
Fig. 6: Evolved SpCas9 variants enable correction of pathogenic SNPs with non-G PAMs.

Data availability

HTS data were deposited in the Sequence Read Archive database (PRJNA596996). Plasmids encoding SpCas9-NRRH, SpCas9-NRTH and SpCas9-NRCH nucleases, BE4 and ABE are available through Addgene. A subset of the selection plasmids used in this study are available through Addgene. Other materials are available from the corresponding authors upon reasonable request.

Code availability

Custom scripts used to analyze the human cell target site library are available at


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We thank T.R. Blum and A. Raguram for helpful discussions. This work was supported by National Institutes of Health grants R01 EB027793, U01 AI142756, RM1 HG009490 and R35 GM118062, the Howard Hughes Medical Institute, the Bill and Melinda Gates Foundation and the St. Jude Collaborative Research Consortium. S.M.M. and M.S. were supported by a National Science Foundation Graduate Research Fellowship. T.W. was supported by a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellowship (F32GM119228). M.A. was supported by an NWO Rubicon Fellowship. G.A.N was supported by a Helen Hay Whitney Postdoctoral Fellowship.

Author information

S.M.M., T.W. and D.R.L. designed the research. S.M.M., T.W. and P.B.R. performed evolution. S.M.M., T.W., P.B.R. and T.P.H. characterized variants in bacteria. S.M.M., T.W., P.B.R., M.A. and Z.M. conducted human cell experiments. M.A. and M.S. designed and constructed the integrated target site human cell library. M.S. wrote custom software. S.M.M., T.W., M.A. and M.S. analyzed library data. S.M.M. and T.W. performed GUIDE-seq experiments. G.N. and H.A.R. designed and performed the sickle cell anemia site experiments. S.M.M., T.W., P.B.R. and T.P.H. designed and performed the HbS editing experiments. S.M.M., T.W. and D.R.L. wrote the manuscript.

Correspondence to David R. Liu.

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Competing interests

The authors declare competing financial interests: S.M.M., T.W. and D.R.L. have filed patent applications on this work. D.R.L. is a consultant and co-founder of Editas Medicine, Pairwise Plants, Beam Therapeutics and Prime Medicine, companies that use genome-editing technologies.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Tables 1, 3 and 6–9, and Notes 1–4

Reporting Summary

Supplementary Table 2

Excel table of PACE mutations.

Supplementary Table 4

Excel table of GUIDE-seq-identified off-target sites.

Supplementary Table 5

Excel table of PAMvar library data on all sites.

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Miller, S.M., Wang, T., Randolph, P.B. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol (2020).

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