The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1,2,3,4,5,6,7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.
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We thank A. Magnell for experimental assistance; R. Macrae for a critical review of the manuscript; and the entire Zhang laboratory for support and advice. D.B.T.C. is supported by T32GM007753 from the National Institute of General Medical Sciences. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. J.C.M. is supported by the NIH (training grant 2 T32 GM 7287-41). H.N. is supported by JST, PRESTO (JPMJPR13L8), JSPS KAKENHI (Grant Numbers 26291010 and 15H01463). O.N. is supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan Agency for Medical Research and Development, AMED, and the Council for Science, and Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology. N.C. is supported by the Karolinska Institutet, the Swedish Research Council (521-2014-2866), the Swedish Cancer Research Foundation (CAN 2015/585), and the Ragnar Söderberg Foundation. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, Howard Hughes Medical Institute, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng.
A patent has been filed relating to the presented data. F.Z. is a founder and scientific advisor for Editas Medicine and a scientific advisor for Horizon Discovery. L.G., D.B.T.C., and F.Z. are co-inventors on US Provisional Patent Application Serial No. 62/324,820, directed to the Cpf1 protein variants, as described in this manuscript.
Integrated supplementary information
Evaluation of (a) single amino acid mutations and (b) combination mutants to construct the AsCpf1 RVR variant, which is active at target sites with TATV PAMs. Dots show mean ± s.e.m. (n = 2).
Histograms of abundances of 48 PAMs (NNNNNNNN) at each in vitro cleavage time point for (a) WT AsCpf1, (b) S542R/K607R, and (c) S542R/K548V/N552R. The color of each histogram represents elapsed time. NNNNVRRT sequences, which were used to center the histograms, are shown in black.
Supplementary Figure 4 (a) Comparison of the activity of WT AsCpf1 to the RR variant at target sites with cytosine-containing PAMs.
(b) Activity of the RR variant at TYCV and VYCV sites (V = A, C, or G), demonstrating that the presence of a 5’ T in the PAM sequence is not always required (i.e., some NYCV PAMs can be recognized). The data for TYCV sites is the same as that shown in (a). All indel percentages were measured in HEK293T cells. Dots show mean ± s.e.m. (n = 2-3).
Supplementary Figure 6 Editing efficiency of the AsCpf1 RR variant at TYCV sites in mouse Neuro2a cells.
(a) Diagram of the mouse PCSK9 locus. Gray boxes represent coding sequences. (b) Indel percentages produced by the RR variant at PCSK9 target sites with TYCV PAMs. Bars show mean ± s.e.m. (n = 3). (c) Representative indels at the target site (#2) with the highest editing efficiency. The red triangle represents the putative cleavage site on the top strand.
(a) Definition of targeting range for Cpf1 and Cas9. Comparison of the targeting range of Cpf1 (+RR and RVR variants) to Cas9 (+VQR and VRER variants) in (b) the human genome and (c) coding sequences. Plots show the probability mass function of the distance (in base pairs) to the nearest cleavage site. The boxplots indicate median and interquartile range. Genomic regions that contain Ns or masked repeats were ignored in this analysis.
Related to Figure 3c. An alanine scan of residues with interactions or putative interactions with the DNA strands. Bars show mean ± s.e.m. (n = 2-3). K949A was selected as a candidate for enhancing the specificity of AsCpf1. Lys949 is part of the bridge helix.
(a) Sequence alignment of 43 Cpf1 or putative Cpf1 orthologs, highlighting the REC1, WED-II, and PI domains, which contain the residues selected for mutagenesis screening. Cpf1 name abbreviations follow conventions we previously reported (Zetsche et al. Cell 2015). (b) Zoom-in of the positions (green boxes) corresponding to the mutated residues in AsCpf1 conferring altered PAM specificity. A red line indicates an insertion of one or more bases in the alignment that are omitted for clarity. See also Supplementary Table 3.
(a) Crystal structures of AsCpf1 (PBD ID: 5B43) and LbCpf1 (PDB ID: 5ID6), highlighting the corresponding residues mutated to alter PAM specificity. The PAM duplex shown for LbCpf1 is a model. (b) Activity of LbCpf1 G532R/K595R and G532R/K538V/Y542R at TYCV and TATV sites, respectively, in HEK293T cells. Each point represents the mean of three replicates, and the red lines indicate the overall means within each group. The data for AsCpf1 also appears in Figure 2e. n.s. p > 0.05 (Mann-Whitney); ****p < 0.0001 (Wilcoxon signed-rank).
Activity of the (a) LbCpf1 RR variant and (b) LbCpf1 RVR variant at target sites with preferred PAMs in HEK293T cells. Dots show mean ± s.e.m. (n = 3). Supplementary Figure 10b shows these data in aggregate. The target sites are the same as those shown in Supplementary Figure 5b-c. For the RR variant, the three CCCC sites are not included in Supplementary Figure 10b.
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Gao, L., Cox, D., Yan, W. et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 35, 789–792 (2017). https://doi.org/10.1038/nbt.3900
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