Abstract
The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2,3,4,5. We also report that four to six bases at the 3′ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.
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Acknowledgements
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., the Jim and Ann Orr Research Scholar Award (to J.K.J.), a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to B.P.K.), an award from the Massachusetts General Hospital Collaborative Center for X-Linked Dystonia-Parkinsonism, and an MGH Tosteson Award (to S.Q.T.). New reagents described in this work have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas).
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B.P.K., S.Q.T., and J.K.J. conceived of and designed experiments. B.P.K., S.Q.T., M.S.P., N.T.N., and M.M.W. performed all human cell experiments and analyzed data. S.Q.T., J.M.L., Z.R.M., and M.J.A. analyzed the GUIDE-seq and targeted deep-sequencing data. B.P.K., S.Q.T., and J.K.J. wrote the manuscript with input from all authors.
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J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Beacon Genomics, Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. JKJ's interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T., M.J.A., and J.K.J. are co-founders of Beacon Genomics, a company that is commercializing methods for determining nuclease specificity.
Integrated supplementary information
Supplementary Figure 1 Cpf1 target sites in endogenous genes known to be efficiently modified by SpCas9.
Sites targeted by Cpf1 and SpCas9 in four genes are highlighted in blue and grey, respectively. Target site sequences and primers used to amplify these loci are provided in Supplementary Tables 1 and 5, respectively.
Supplementary Figure 2 On-target time-course activity assays of AsCpf1, LbCpf1, and SpCas9.
Time-course assays were performed using guide RNAs targeted to matched sites in human cells. Percent modification activity determined by T7E1 assay; error bars, s.e.m.; n = 3.
Supplementary Figure 3 Cpf1 activity and specificity with alternate length crRNAs.
(a) Indel mutation percentages induced by AsCpf1 and LbCpf1 at three different endogenous sites using crRNAs with variable length 3’ end truncations, determined by T7E1 assay. Error bars, s.e.m.; n = 3; nt, nucleotide. (b) Endogenous gene modification by AsCpf1 and LbCpf1 using crRNAs with 20 nt spacers that contain singly mismatched bases. Activity determined by T7E1 assay; error bars, s.e.m.; n = 3.
Supplementary Figure 4 Tag integration and overall mutagenesis percentages observed in GUIDE-seq experiments in U2OS cells.
(a,b) On-target GUIDE-seq tag integration (a) and overall mutagenesis (b) determined by RFLP and T7E1 assays, respectively, for the 11 matched target sites examined by GUIDE-seq in Figure 3b. Error bars, s.e.m.; n = 3. (c) Ratios of tag integration to overall mutagenesis (from data in a and b) for the 11 matched target sites. (d) Summary of RFLP:T7E1 ratios from c, with means and 95% confidence intervals shown. (e,f) On-target GUIDE-seq tag integration (e) and overall mutagenesis percentages (f) determined by RFLP and T7E1 assays, respectively, for the 9 different endogenous human gene target sites examined by GUIDE-seq in Figure 3b. Error bars, s.e.m.; n = 3. (g) Summary of RFLP:T7E1 ratios calculated from the data in e and f, with means and 95% confidence intervals shown.
Supplementary Figure 5 Genome-wide specificities of AsCpf1 and LbCpf1 using the crRNA targeted to matched site 6.
Off-target sites were determined using GUIDE-seq. Mismatched positions in the target sites of off-targets are highlighted in color, and GUIDE-seq read counts shown to the right of the on- and off-target sequences represent a measure of cleavage efficiency at a given site.
Supplementary Figure 6 GUIDE-seq with AsCpf1 and LbCpf1 in HEK293 cells.
(a,b) On-target GUIDE-seq tag integration (a) and overall mutagenesis (b) determined by RFLP and T7E1 assays, respectively, for 6 crRNAs. Error bars, s.e.m.; n = 3. (c) Ratios of tag integration to overall mutagenesis (from data in a and b), with means and 95% confidence intervals shown. (d) Off-target sites for AsCpf1 and LbCpf1 with three different crRNAs in HEK293 and U2OS cells determined using GUIDE-seq (U2OS cell data is re-represented from Figure 3b for comparative purposes). Mismatched positions in the target sites of off-targets are highlighted in color, and GUIDE-seq read counts shown to the right of the on- and off-target sequences represent a measure of cleavage efficiency at a given site.
Supplementary Figure 7 Deep sequencing of on-target and closely mismatched potential Cpf1 off-target sites.
Triplicate data points representing percent modification are plotted for control, AsCpf1, and LbCpf1 experiments; if less than 3 data points are shown for any given condition, the value is either zero or not determined (see Supplementary Table 4). Mismatched nucleotides in off-target sites are colored; off-target sites are numbered as shown in Supplementary Tables 4 or 5; on, on-target site; Gs, off-target site detected in GUIDE-seq experiments shown in Fig. 3b and Supplementary Fig. 5; Ss, statistically significant indel formation above background control levels determined using the Bonferroni correction (Supplementary Table 4).
Supplementary Figure 8 Venn diagrams of Cpf1 and SpCas9 targeting ranges based on availability of sites in the reference human genome.
(a, b) Targetable genetic elements are defined as those that contain the predicted cleavage site of at least one Cpf1 or SpCas9 target site, without or with a polIII promoter required 5’-guanine for SpCas9 (panels a and b, respectively; within N21GG versus TTTN24 motifs or GN20GG versus TTTN24 motifs, respectively). Numbers in the bottom right of each plot represent genetic elements not targetable by either SpCas9 or Cpf1. TSS, transcription start site.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Note 1 (PDF 1741 kb)
Supplementary Table 1
gRNA target sites. (XLSX 24 kb)
Supplementary Table 2
In Silico off-targets. (XLSX 63 kb)
Supplementary Table 3
Deep sequencing data and p-values. (XLSX 268 kb)
Supplementary Table 4
GUIDE-seq data. (XLSX 79 kb)
Supplementary Table 5
Oligonucleotides. (XLSX 19 kb)
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Kleinstiver, B., Tsai, S., Prew, M. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34, 869–874 (2016). https://doi.org/10.1038/nbt.3620
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DOI: https://doi.org/10.1038/nbt.3620
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