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. 2017 Aug;35(8):789-792.
doi: 10.1038/nbt.3900. Epub 2017 Jun 5.

Engineered Cpf1 variants with altered PAM specificities

Linyi Gao  1   2 , David B T Cox  1   3   4 , Winston X Yan  1   4   5 , John C Manteiga  3 , Martin W Schneider  1 , Takashi Yamano  6 , Hiroshi Nishimasu  6   7 , Osamu Nureki  6 , Nicola Crosetto  8 , Feng Zhang  1   2   9   10
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Engineered Cpf1 variants with altered PAM specificities

Linyi Gao et al. Nat Biotechnol. .
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Abstract

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. 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 BLISS 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.

Figures

Figure 1
A bacterial interference-based negative selection screen identifies amino acid substitutions of AsCpf1 conferring activity at non-canonical PAMs. (a) Crystal structure of AsCpf1 (PDB ID: 5B43) in complex with crRNA and target DNA, highlighting the PAM nucleotides (magenta), and PAM-proximal residues selected for mutagenesis (blue). (b) Schematic of bacterial interference assay used to identify variants with altered PAM specificity. (c) Sensitivity of wild-type AsCpf1 to substitution mutations in the PAM as measured by bacterial interference. Bars show mean ± s.e.m. of n = 3 plated transformations. (d) Scatter plots of screen readout, highlighting depleted variants. Each dot represents a distinct wild-type or mutant codon. The dashed line indicates 15-fold depletion.
Figure 2
Construction and characterization of AsCpf1 variants with altered PAM specificities. (a) Combinatorial mutagenesis identifies AsCpf1 variants that cleave target sites with TYCV and TATV PAMs in HEK293T cells, where Y = C or T, and V = A, C, or G (see also Supplementary Fig. 1). Bars show mean ± s.e.m. for n = 4 transfected cell cultures. (b) Schematic of in vitro cleavage assay used to determine global PAM specificity (see also Supplementary Fig. 2–3). (c) Web logos of the most rapidly cleaved PAMs for wild-type (WT), S542R/K607R (RR), and S542R/K548V/N552R (RVR) variants. (d) Normalized cleavage rates for all 4-base PAMs for WT and variants. NNRN PAMs are not shown due to negligible cleavage. The most active PAMs are boxed in red. (e) Comparison of the activity of WT, RR, and RVR at their preferred PAMs at a diverse panel of target sites in HEK293T cells (see also Supplementary Fig. 5). For indel percentages, each dot represents the mean of n = 3 transfected cell cultures, and the red lines indicate the overall means within each group. For fold improvement, each dot represents the ratio of the means of the corresponding indel replicates. n.s. p > 0.05 (Mann-Whitney); *p < 0.05 (Mann-Whitney); ****p < 0.0001 (Wilcoxon signed-rank). (f) Targeting range of AsCpf1 variants in the human genome and in coding sequences (see also Supplementary Fig. 7). 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.
Figure 3
Specificity of AsCpf1 PAM variants. (a) DNA double-strand breaks labeling in situ and sequencing (BLISS) for 4 target sites (VEGFA, GRIN2B, EMX1, and DNMT1) in HEK293T cells. The log10 number of unique double-strand break (DSB) ends per 105 reads is indicated by the magenta heat map. The normalized PAM cleavage rates from the in vitro cleavage assay in Fig. 2d are indicated by the blue heat map. Each BLISS-identified cleavage site was independently assessed for indel formation (bar graphs). Bars show mean ± s.e.m. for n = 4 transfected cell cultures. Mismatches in bases 21–23 of the target are grayed as they have minimal impact on cleavage efficiency, . (b) Evaluation of an additional target site in the RPL32P3 gene with known TTTV off-target sites. (c) Addition of a K949A mutation improves the specificity of WT AsCpf1 and variants (see also Supplementary Fig. 8). For (b) and (c), bars show mean ± s.e.m. for n = 3 transfected cell cultures. (d) On-target efficiency of the RR and RVR variants ± K949A. Each dot represents the mean of n = 3 transfected cell cultures.
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