Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells - PubMed (original) (raw)

Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

Benjamin P Kleinstiver et al. Nat Biotechnol. 2016 Aug.

Abstract

The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases 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). 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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Figures

Figure 1

Figure 1

On-target indel mutation percentages induced by Cpf1 nucleases in human cells. (a) Endogenous gene percent modification induced by AsCpf1 and LbCpf1 at 19 endogenous human gene sites (left panel, determined by T7E1 assay). Target sites were chosen in amplicons known to be efficiently modified by SpCas9 (right panel, and see Supplementary Fig. 1). Error bars, s.e.m.; n = 3. (b) and (c) Matched target sites for AsCpf1, LbCpf1, and SpCas9 that share a common protospacer sequence (panel b) were examined for mutagenesis by AsCpf1, LbCpf1, and SpCas9 nucleases (determined by T7E1 assay; panel c). Error bars, s.e.m.; n = at least 3; ND, not detected. (d) Summary of matched site on-target activities from panel c, with means and 95% confidence intervals shown.

Figure 2

Figure 2

Tolerance of AsCpf1 and LbCpf1 to mismatched or truncated crRNAs. (a, b) Endogenous gene modification by AsCpf1 and LbCpf1 using crRNAs that contain pairs of mismatched bases (panel a) or singly mismatched bases (panel b). Activity determined by T7E1 assay; error bars, s.e.m.; n = 3. (c) Summary of mutagenesis percentages by AsCpf1 and LbCpf1 at 3 different endogenous sites with crRNAs bearing variable length 3’ end truncations or extensions (see also Supplementary Fig. 3a), where activities are normalized to mutagenesis observed when using the canonical 23 nucleotide spacer. Error bars, s.e.m.; n = 3; nt, nucleotide. (d) Activity of AsCpf1 and LbCpf1 when programed with singly mismatched crRNAs either of 23 or 20 nucleotides in length. For clarity, data from panel b is re-presented here. Activity determined by T7E1 assay; error bars, s.e.m.; n = 3.

Figure 3

Figure 3

Genome-wide specificities of AsCpf1 and LbCpf1. (a) Relative GUIDE-seq read start mapping position. The first 5’ base of the protospacer adjacent to the PAM is position 1. Reads that start within the protospacer region are colored in blue, reads that originate outside are colored in red. Reads mapping to the plus strand are depicted above the x-axis, reads mapping in the reverse orientation are depicted below. (b) Off-target sites for AsCpf1 and LbCpf1 with 20 different crRNAs, determined using GUIDE-seq in U2OS cells. 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. (c) and (d) Summary of the total number of off-target sites detected by GUIDE-seq in U2OS cells with 20 crRNAs (panel c) and in HEK293 cells with 3 crRNAs (panel d). The off-target sequences and GUIDE-seq read counts for matched site #6 are shown in Supplementary Fig. 5. (e) Scatterplots of mean mutagenesis versus GUIDE-seq read counts analyzed from independent samples. Mutagenesis percentages for AsCpf1 that are not significantly different from negative controls are indicated with triangles. Error bars, s.e.m.; n = 3.

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References

    1. Zetsche B, et al. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015;163:759–771. - PMC - PubMed
    1. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32:347–355. - PMC - PubMed
    1. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278. - PMC - PubMed
    1. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096. - PubMed
    1. Maeder ML, Gersbach CA. Genome-editing Technologies for Gene and Cell Therapy. Mol Ther. 2016 - PMC - PubMed

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