Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing

Abstract

Broad use of CRISPR–Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.

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Fig. 1: Engineering and characterization of AsCas12a variants with expanded target range in human cells.
Fig. 2: AsCas12a variants enhance on-target editing in human cells.
Fig. 3: Improved multiplex editing, gene activation and base editing with enAsCas12a.
Fig. 4: Characterization and improvement of AsCas12a specificity and activity.

Code availability

The custom Python script for PAMDA data analysis and the PAM identification and enumeration script will be made available upon request.

Data availability

Data sets from GUIDE-seq and high-throughput sequencing experiments (for PAMDA and base editing experiments) have been deposited with the National Center for Biotechnology Information Sequence Read Archive under BioProject ID PRJNA508751.

Change history

  • 15 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank J. Grünewald for preparing paramagnetic beads, S. Garcia for informatics support and members of the Joung and Maus laboratories for advice. This work was supported by the Desmond and Ann Heathwood MGH Research Scholar Award (to J.K.J.), Natural Sciences and Engineering Research Council of Canada (NSERC) Banting and Charles A. King Trust Postdoctoral Fellowships (B.P.K.), the National Institutes of Health (NIH) Awards nos K99 CA218870 (B.P.K.), R00 HG008399 (L.P.), R35 GM118158 (M.J.A. and J.K.J.), and RM1 HG009490 (J.K.J.), the Bill and Melinda Gates Foundation grant no. OPP1159968 (J.K.J.) and an award from the Massachusetts General Hospital Collaborative Center for X-Linked Dystonia-Parkinsonism (J.K.J.). Plasmids described in this work are available through the nonprofit plasmid repository Addgene (http://www.addgene.org/crispr-cas).

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Authors

Contributions

B.P.K., A.A.S., R.T.W. and J.K.J. conceived of and designed the experiments. Experiments were performed by B.P.K., A.A.S., R.T.W., Y.E.T., M.M.W. and J.E.H. Data sets from PAMDA, deep sequencing and GUIDE-seq experiments were analyzed by B.P.K., A.A.S., R.T.W., J.Y.H., K.C., J.M.-L., L.P. and M.J.A. I.S. and M.V.M. provided expertise for experiments in primary T cells. The manuscript was written by B.P.K. and J.K.J. with input from all authors.

Corresponding authors

Correspondence to Benjamin P. Kleinstiver or J. Keith Joung.

Ethics declarations

Competing interests

B.P.K. is a scientific advisor to Avectas. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.K.J. has financial interests in Beam Therapeutics, Blink Therapeutics, Editas Medicine, Endcadia, Monitor Biotechnologies (formerly known as Beacon Genomics), Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. M.J.A. has financial interests in Monitor Biotechnologies. J.K.J. holds equity in EpiLogic Therapeutics. J.K.J.’s and M.J.A.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. B.P.K., A.A.S., and J.K.J. are inventors on patent applications covering the Cas12a variants described in this work. Y.E.T., B.P.K. and J.K.J. are inventors on a patent application describing Cas12-based transcriptional activators. M.V.M. reports personal fees from Adaptimmune, personal fees from Adaptive Biotechnologies, grants and personal fees from Agentus, personal fees from Bluebird Bio, personal fees from Cellectis, grants and personal fees from CRISPR Therapeutics, personal fees from Incysus, grants and personal fees from Kite Pharma, personal fees from Juno, personal fees from MPM, personal fees from Novartis, personal fees from Takeda, grants and personal fees from TCR2 (SAB), personal fees from Third Rock Ventures, personal fees from Windmil (SAB) and personal fees from Century, outside the submitted work. M.V.M. has a patent related to CAR T cells for multiple myeloma, lymphoma and glioblastoma (none of which are the subject of this manuscript) pending.

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Integrated supplementary information

Supplementary Figure 1 Activities of Cas12a orthologs and engineered variants in human cells.

(a) Activities of Cas12a orthologs targeted to endogenous sites in human cells bearing TTTN or VTTN PAMs. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. (b) Summary of the activities of Cas12a orthologs against 24 sites with NTTN PAM sequences (mean activities shown from data in panel a). *, P < 0.05; ****, P < 0.0001 (Mann–Whitney, two-tailed; P values in Supplementary Table 8). (c) Schematic and structural representations of Cas12a paired with a crRNA, and interacting with a target site encoding a prototypical TTTA PAM. In structural representations, amino acid residues proximal to PAM DNA bases are highlighted in green; images generated from PDBID:5B43 (ref. 17) visualized in PyMOL (v 1.8.6.0); select regions of the PAM-interacting domain are hidden for clarity. (d, e) Activities of AsCas12a variants bearing single amino acid substitutions when tested against endogenous sites in human cells bearing canonical (panel d) or non-canonical (panel e) PAMs. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. (f) Modification of endogenous sites in human cells by wild-type AsCas12a and variants bearing amino acid substitutions. Activities assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. Reference. 17. Yamano, T. et al. Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell 165, 949–962 (2016).

Supplementary Figure 2 Optimization of an in vitro PAM determination assay.

(a) Representative SDS–PAGE gel images of purified Cas12a orthologs and variants; s.m, size marker in kDa. Each protein was purified and analyzed by SDS–PAGE once. (b) Schematic of linearized plasmid bearing combinations of PAMs and spacers used as substrates for in vitro cleavage reactions. (c) Time-course in vitro cleavage reaction profiles of wild-type AsCas12a (left panel) and the E174R/S542R/K548R variant (right panel) on the substrates illustrated in panel b. Curves were fit using a one phase exponential decay equation; mean and error bars represent s.e.m for n= 3. (d) Schematic of the PAM determination assay (PAMDA). Linearized plasmid libraries harboring 10 randomized nucleotides in place of the PAM were subjected to in vitro cleavage reactions with Cas12a ribonucleoprotein (RNP) complexes. Aliquots of the reaction were stopped at various time points and subsequently used as template for PCR. Substrates harboring incompletely targetable PAMs were amplified and sequenced to enable quantification of the rate of PAM depletion from the starting library over time. (e) Correlation between PAMDA rate constants (k) across replicates of wild-type AsCas12a (left panel) and the E174R/S542R/K548R variant (right panel). (f) Correlation between rate constants from mean PAMDA values across two spacer sequences. (g) Histogram of PAMDA rate constants for wild-type and E174R/S542R/K548R AsCas12a. (h) Depletion profiles over time of substrates from the PAMDA encoding the indicated PAM sequences. Curves were fit using a one phase exponential decay equation; mean and error bars represent s.e.m for n= 4. (i) Comparison of the PAM preference profiles of the E174R/S542R and E174R/S542R/K548R variants across all 128 NNYN PAMs (re-plotted from Fig. 1b; Y = C or T). The gray shaded box indicates an arbitrary PAMDA rate constant threshold of 0.005 (or 10−2.25) roughly predictive of activity in human cells (see Supplementary Fig. 3g); mean and error bars represent s.e.m for n= 4.

Supplementary Figure 3 Assessment of the improved targeting range of enAsCas12a in human cells.

(a, b) Comparison of the activities of E174R/S542R and E174R/S542R/K548R AsCas12a on endogenous sites in human cells bearing non-canonical VTTN and TTCN PAMs (panel a), or TATN PAMs (panel b). (c) Comparison of the activities of wild-type, E174R/S542R, and E174R/S542R/K548R AsCas12a on sites with TTTT PAMs. (d) Activity of wild-type AsCas12a on sites with TTCN or TATN PAMs. (e, f) Activity of the E174R/S542R/K548R variant against sites with TGTV PAMs (panel e) or additional sites with various non-canonical PAMs (panel f). (g) Correlation between the PAMDA rate constant and mean modification in human cells for the PAMs tested in panels a-e. The gray shaded box indicates an arbitrary PAMDA rate constant threshold of 0.005 (or 10−2.25) roughly predictive of activity in human cells. (h) Summary of targetable PAMs for enAsCas12a. Tiers of PAMs: 1, high-confidence PAM (mean k > 0.01, mean percent modified > 20%); 2, medium confidence PAM (mean k > 0.005, mean percent modified > 10%); 3, low activity or discrepant PAM (mean percent modified < 10% or discrepancy between mean k and percent modified). See also Supplementary Table 2. (i) Influence of the +1 (most PAM proximal) base identity on the activities of wild-type AsCas12a and enAsCas12a when targeting TTTN PAMs. The mean activities of the 26 TTTN PAM sites from Supplementary Figs. 3c and 4a are shown, with black bars representing the mean; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8); **, P < 0.01 (Wilcoxon signed-rank, two-tailed; P values in Supplementary Table 8). (j) Influence of the +1 base identity on the activity of enAsCas12a when targeting sites with non-canonical TTTT, VTTV, TTCN, and TRTV PAMs. The mean activities of the 92 sites from Supplementary Figs. 3a-c, and 3e are shown, with black bars representing the mean; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8). (k) Impact of percent GC content on the activity of enAsCas12a when targeting sites bearing canonical or non-canonical PAMs (TTYN, VTTV, and TRTV). The mean activities of the 113 sites from Supplementary Figs. 3a-c, 3e, and 4a are binned according to GC content and shown in this panel as box and whisker plots in gray (min, max, median, and quartiles shown), and black bars representing the mean. (l) Sequence logos of targets sites (8 nt PAM and 28 nt spacer) examined with enAsCas12a binned based on mean percent modification. Activities against sites with canonical or non-canonical PAMs (TTYN, VTTV, and TRTV) are from Supplementary Figs. 3a-c, 3e, and 4a. (m) PAMDA data to examine potential -5 PAM position preference exhibited by wild-type AsCas12a or enAsCas12a. (n) Mean modification activity for enAsCas12a grouped based on -5 PAM base identity. Activities from 87 sites with non-canonical NVTTV, NTTCN, and NTRTV PAMs (from Supplementary Figs. 3a, b, and 3e) are shown, with black bars representing the mean of that group; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8).

Supplementary Figure 4 Enhanced on-target activities of Cas12a variants.

(a) Activities of wild-type AsCas12a, E174R/S542R, and enAsCas12a on sites with TTTV PAMs. (b-d) Comparison of the endogenous site modification activities of AsCas12a variants on sites with TTTN PAMs (panel b), TATN PAMs (panel c), and TYCN PAMs (panel d). Percent modification for panels a-d assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. (e) PAM preference profiles for original and enhanced RVR and RR AsCas12a variants assessed by PAMDA. The log10 rate constants are the mean of four replicates, two each against two distinct spacer sequences (see Supplementary Fig. 2d). (f) Comparison of the PAM preference profiles of enAsCas12a and enAsCas12a-HF1 across all 128 NNYN PAMs (see Supplementary Table 1; Y = C or T). The only PAMs targetable by wild-type AsCas12a are of the TTTV class (Fig. 1b). The gray shaded box indicates an arbitrary PAMDA rate constant threshold of 0.005 (or 10−2.25) roughly predictive of activity in human cells (see Supplementary Fig. 3g); mean and error bars represent s.e.m. for n= 4. AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; RVR, S542R/K548V/N552R; enRVR, E174R/S542R/K548V/N552R; RR, S542R/K607R; enRR, E174R/S542R/K607R.

Supplementary Figure 5 enAsCas12a increases potency and expands targeting range of epigenetic editing fusions.

(a) Schematic of VPR activation domain fusions to DNase-inactive Cas12a (dCas12a) orthologs and variants. VPR, synthetic VP64-p65-Rta activation domain26; NLS(sv), SV40 nuclear localization signal; NLS(nuc), nucleoplasmin nuclear localization signal; gs, glycine-serine peptide linker; HA, Human influenza hemagglutinin tag. (b) Illustration of the sequence window encompassing ~700 bp upstream of the VEGFA transcription start site (TSS), with target sites for SpCas9 and Cas12a indicated. (c, d) Activities of dCas12a–VPR architectures in HEK293 cells as judged by VEGFA activation using pairs of crRNAs targeted to sites with TTTV PAMs (panel c) or TTCV PAMs (panel d) in the VEGFA promoter (see panel b). In panel c, SpCas9–VPR using pairs of previously described sgRNAs48 were used as a positive control. Activities assessed via changes in VEGFA production compared to a control transfection containing enAs–VPR(1.3) and a mock crRNA plasmid (bkgd); mean, s.e.m., and individual data points shown for n= 4. (e, f) VEGFA activation by dCas12a–VPR(1.1) or dSpCas9–VPR fusion proteins in HEK293 cells using pools of three or two (panels e and f, respectively) crRNAs or sgRNAs across a range of sites with canonical and non-canonical PAMs for the dCas12a–VPR fusions; mean, s.e.m., and individual data points shown for n= 3. References:26. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015). 48. Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

Supplementary Figure 6 Properties of base editors derived from enAsCas12a and Cas12a orthologs.

(a) Schematic of dCas12a base-editor (BE) constructs with varying NLS and linker compositions. NLS(sv), SV40 nuclear localization signal; NLS(nuc), nucleoplasmin nuclear localization signal; gs, glycine-serine peptide linker; rAPO1, rat APOBEC1; UGI, uracil glycosylase inhibitor. (b) Fold-change in cytosine to thymine (C-to-T) editing compared to the untreated control across all Cs in the 20 nt spacers of 8 target sites. (c) Influence of identity of the preceding (5’) base on C-to-T editing efficiency across eight target sites (see Fig. 3h) is plotted for all Cs in the window encompassing the -14 to +30 region of each target site (an additional 10 nt upstream of the 4 nt PAM and 10 nt downstream of the 20 nt spacer sequence; see Supplementary Table 3). (d) Analysis of edit purity at six selected cytosines across five target sites. The fraction of each non-C identity is plotted over the sum of all non-C occurrences at that position for each BE construct. Bkgd, distribution of nucleotide substitutions or deletions in control samples. Mean and s.e.m. shown for n= 3. (e) Percent insertion or deletion mutation (indel) across sites targeted with dCas12a-BEs. Indels were calculated for each BE/crRNA pair by determining the percentage of alleles encoding an indel within the -14 to +30 window, not counting alleles with substitutions only. Mean and s.e.m. shown for n= 3.

Supplementary Figure 7 Genome-wide specificity assessment of AsCas12a and AsCas12a variants.

(a) Schematic of the GUIDE-seq method31. (b, c) Comparison of the on-target mutagenesis (panel b) and GUIDE-seq dsODN tag integration (panel c) activities of AsCas12a nucleases for GUIDE-seq samples. Percent modification and tag integration assessed by T7E1 and RFLP assays, respectively; mean, s.e.m., and individual data points shown for n= 3. (d) Ratio of GUIDE-seq dsODN tag integration to overall mutagenesis for AsCas12a nucleases; data from panels b and c, mean, s.e.m., and individual data points shown for n= 3. (e, f) GUIDE-seq genome-wide specificity profiles for AsCas12a, enAsCas12a, and enAsCas12a-HF1 each paired with crRNAs targeting sites with TTTV PAMs (panel e) or non-canonical PAMs (panel f). Mismatched positions in off-target sites are highlighted in color; GUIDE-seq read counts are shown to the right of the sequences; yellow diamonds indicate off-target sites that are only supported by asymmetric GUIDE-seq reads; green circles indicate off-target sites previously identified10 for LbCas12a; alternate nucleotides in non-canonical PAMs with mean PAMDA ks > 0.005 for enAsCas12a are not colored/highlighted as mismatches; enAsCas12a-HF1 not assessed on CTTA-1, CTTC-2, or TATC-1. AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R. References:10. Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR–Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016). 31. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

Supplementary Figure 8 Assessment and improvement of AsCas12a specificity.

(a) Activities of wild-type AsCas12a or variants bearing single substitutions when using crRNAs that perfectly match the on-target site or that encode single nucleotide mismatches. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. (b) Activities of enAsCas12a variants bearing additional single amino acid substitutions, assessed as in panel a. (c) PAM preference profiles of enAsCas12a and enAsCas12a-HF1 assessed by PAMDA. The log10 rate constants are the mean of four replicates, two each against two distinct spacer sequences (see Supplementary Fig. 2d). (d) Comparison of the PAM preference profiles of enAsCas12a and enAsCas12a-HF1 across all 128 NNYN PAMs (Y = C or T). The gray shaded box indicates an arbitrary PAMDA rate constant threshold of 0.005 (or 10−2.25) roughly predictive of activity in human cells (see Supplementary Fig. 3g); mean and error bars represent s.e.m for n= 4. (e) Scatterplot of the PAMDA determined rate constants for each NNNN PAM to compare the PAM preferences of enAsCas12a and enAsCas12a-HF1. AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R.

Supplementary Figure 9 On-target activity assessment of AsCas12a, enAsCas12a, and enAsCas12a-HF1.

(a) Time-course in vitro cleavage reactions of Cas12a orthologs and variants conducted at 37, 32, and 25 °C (left, middle, and right panels, respectively) using the PAMDA site 1 substrate. Curves were fit using a one phase exponential decay equation; mean and error bars represent s.e.m for n= 3. (b, c) Assessment of the on-target activities of AsCas12a, enAsCas12a, and enAsCas12a-HF1 by plasmid electroporation into U2OS cells on target sites harboring TTTV PAMs (panel b) or non-canonical VTTV, TATV, TTCV, and TTTT PAMs (panel c). Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3. (d, e, f) Assessment of the on-target activities of AsCas12a, enAsCas12a, and enAsCas12a-HF1 when delivered as RNPs into HEK2923 cells (panels d and e) and primary T cells (panel f). The mean activities for each site in panel d are shown in panel e. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n= 3; ns, P > 0.05; *, P < 0.05 (Wilcoxon signed-rank, two-tailed; P values in Supplementary Table 8). AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Results, Supplementary Discussion, Supplementary Sequences

Reporting Summary

Supplementary Table 1

PAMDA data

Supplementary Table 2

Classification of PAMs targetable by AsCas12a variants

Supplementary Table 3

Base editing data

Supplementary Table 4

GUIDE-seq data

Supplementary Table 5

Plasmid list

Supplementary Table 6

crRNA and sgRNA list

Supplementary Table 7

Oligonucleotide list

Supplementary Table 8

P values

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Kleinstiver, B.P., Sousa, A.A., Walton, R.T. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol 37, 276–282 (2019). https://doi.org/10.1038/s41587-018-0011-0

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