Abstract
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR–Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.
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Acknowledgements
This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-059680) to Y.D. and the Canadian Institutes of Health Research (CIHR; MOP-84260) to G.D. Salary support was provided by the Fonds de la Recherche du Québec-Santé (FRQS) to D.A. and Y.D. and by a Bridge Grant from the Beatrice Hunter Cancer Research Institute (BHCRI) to J.S. Partial salary support to L.B. was obtained through the Mitacs Accelerate program. We thank the staff at the Hôpital St-Francois d'Assise for their assistance in collecting UCB units, M.-È. Rhéaume for processing and HSPC purification, and our colleagues for critical reading of the manuscript. We thank A. Fradet-Turcotte (Centre de Recherche du CHU de Québec–Université Laval), G. Church (Department of Genetics, Harvard Medical School), and F. Zhang (Broad Institute of MIT and Harvard) for providing materials.
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Conceptualization, Y.D.; methodology, D.A., A.D., L.B., C.C.H., S.C., J. Laganière, and Y.D.; investigation, D.A., A.D., L.B., C.C.H., S.C., J. Loehr, D.S., M.D., and Y.D.; resources, J.S. and G.D.; writing original draft, Y.D.; writing, review and editing, D.A., A.D., L.B., S.C., G.D., J. Laganière, and Y.D.; supervision, J. Laganière and Y.D.; funding acquisition, J. Laganière and Y.D.
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A provisional patent application has been filed with the United States Patent and Trademark Office in relation to this work.
Integrated supplementary information
Supplementary Figure 1 ATP1A1 variants with in-frame deletions are enriched in ouabain-resistant cell populations.
(a) Total editing efficacy along with spectrum and frequency of individual indels as determined by the TIDE assay in K562 cells treated with ATP1A1 G2 sgRNA. Analysis was performed on genomic DNA samples shown in Figure 1b. (b) Same as (a) but for ATP1A1 G4 sgRNA. (c) Same as (a) but for ATP1A1 G3 sgRNA.
Supplementary Figure 2 HDR-driven editing at ATP1A1 induces cellular resistance to ouabain.
(a) Schematic representation of the intronic SpCas9 target site G3 and partial sequences of single-stranded oligodeoxynucleotides (ssODNs) donors used to introduce the Q118D/R and N129D/R mutations. Annotated are novel restriction sites to monitor the insertion of ssODN-specified mutations. (b) K562 cells stably expressing SpCas9 were co-transfected with an ATP1A1 G3 sgRNA expression vector (500ng) along with the indicated ssODNs (10pmol). Genomic DNA was harvested 10 days post-transfection and a PvuI RFLP assay was used to determine the frequency of HDR at the cleavage site, indicated as the % HDR at the base of each lane. Where indicated, cells were treated with 0.5μM ouabain for 7 days starting 3 days post transfection. An expression vector encoding EGFP (-) was used as a negative control. (c) Same as in (b) but using BmgBI. (d) Same as in (b) but using ClaI. (e) Same as in (b) but using Hpy188I. (f) Same as in (e) but cells initially selected at 0.5μM ouabain were cultivated in the presence of increasing concentrations of the drug for a week. (g) Uncropped gel images for panels (e) and (f). A naturally occurring Hpy188I (TCNGA cleavage site) is present in the amplicon creating the strong RFLP signal indicated by the red arrows. The HDR-specific signal (black arrow; predicted at 77bp) is running below the 100bp marker and is best viewed when the gels are overexposed making it is impossible to quantify this signal using RFLP-based assays.
Supplementary Figure 3 Ouabain-based selection is portable to various cell lines and compatible with CRISPR–AsCpf1-driven editing.
(a) U2OS cells were treated and analyzed as in Figure 1b. (b) hTERT-RPE-1 cells were treated and analyzed as in Figure 1b and Supplementary Figure 2d. (c) Schematic representation of AsCpf1 target sites surrounding DNA encoding the first extracellular loop of human ATP1A1. Annotated are the positions of residues Q118 and N129, exon/intron boundary, protospacer adjacent motifs (PAM) and five potential AsCpf1 target sequences (Targets 1-5). Also shown are partial sequences of single-stranded oligodeoxynucleotides (ssODNs) donors used to introduce the Q118R and N129D mutations. Annotated are novel restriction sites to monitor the insertion of ssODN-specified mutations. (d) K562 cells stably expressing AsCpf1 were transfected with ATP1A1 crRNA expression vectors (G1-G5) (500 ng) and the Surveyor assay was used to determine the frequency of AsCpf1-induced insertions and deletions, indicated as the % Indels at the base of each lane. Where indicated, cells were treated with 0.5μM ouabain for 7 days starting 3 days post transfection. An expression vector encoding EGFP (-) was used as a negative control. (e) K562 cells were transfected with a vector expressing eSpCas9(1.1) and the ATP1A1 G3 sgRNA (500ng each) or with a vector expressing AsCpf1 and the ATP1A1 G5 crRNA (500ng each) (see Supplementary Figure 12 for details on the vectors) along with the indicated ssODNs. Cells were treated as in (d) and assayed using BmgBI and EcoRI RFLP assays.
Supplementary Figure 4 Selection for CRISPR–Cas9-driven targeted mutagenesis by coediting ATP1A1 via NHEJ.
(a) Experimental strategy for the co-enrichment of CRISPR-driven editing at a second locus. GOI, gene of interest. (b) Typical selection process. Timing can vary according to initial modification rates at ATP1A1. (c) Schematic of the dual eSpCas9(1.1) and tandem U6-driven sgRNAs expression vector (see also Supplementary Figure 12). (d) Surveyor assays to determine on-target and off-target activity for the EMX1 sgRNA on samples reported in Table 1. (e) Same as (d) but for co-targeting AAVS1. (f) Surveyor assays on samples from K562 cells transiently co-transfected with WT SpCas9 and sgRNA expression vectors targeting AAVS1 (0.5μg and 1μg of each vector).
Supplementary Figure 5 Selection for CRISPR–AsCpf1-driven targeted mutagenesis by coediting ATP1A1 via NHEJ.
(a) Schematic of the dual AsCpf1 and U6-driven crRNA array expression vector (see also Supplementary Figure 12). (b) K562 cells were transfected with 500ng of the vectors shown in (a), treated and assayed for indels as shown in Supplementary Figure 4d. (c) Same as in (a). (d) Same as in (b) but using 1μg of the vector. (e) HEK293 cells were treated and analyzed as in (b) but the TIDE assay was used to determine the frequency of indels. Value for ATP1A1 relates to the co-selection performed with the DNMT1 crRNA. (f) Same as in (e) but using the Surveyor assay.
Supplementary Figure 6 Off-target analysis in K562 and HEK293 cells coselected with the AsCpf1 crRNA array vector targeting DNMT1.
(a) Schematic of the dual AsCpf1 and U6-driven crRNA array expression vector. (b) Surveyor assays to determine on-target and off-target activity performed on samples reported in Table 1 and Supplementary Table 1. To facilitate the interpretation of the results, the gels for on-target activity presented in Supplementary Figure 5b,f have been reproduced here. Off-target activity at DNMT1-OT1 was assayed using two different sets of primers since it is found within a repetitive element making its detection more challenging. (c) On-target activity at DNMT1 was also determined using the TIDE assay in both co-selected and transiently transfected cells. (d) Surveyor assays to determine on-target and off-target activity in K562 cells transiently transfected with 1μg and 2μg of the vector shown in (a).
Supplementary Figure 7 Selection for CRISPR-driven targeted integration at LMNA by coediting ATP1A1 via HDR.
(a) Targeting scheme for the integration of Clover and mRuby2 to the N-terminus of Lamin A/C. (b) Fluorescence imaging of ouabain-treated cells expressing the Lamin A/C-Clover fusion. Scale bar, 25μm (c) A ClaI RFLP assay was used to determine the frequency of SpCas9-induced HDR at the ATP1A1 locus in samples shown in Figure 2. % HDR is indicated at the base of each lane. (d) FACS-based quantification of Clover targeting to the N-terminus of Lamin A/C at various doses of WT SpCas9 and eSpCas9(1.1) in presence or absence of co-selection with ouabain. (e) Same as in (d) but in cells transfected with both Clover and mRuby2 donors. (f) Same as in (d) but targeting LMNA using an AsCpf1 crRNA array. Transfection conditions are indicated in Supplementary Table 2.
Supplementary Figure 8 Sequencing of non-HDR alleles from LMNA coselection experiments.
(a) Schematic of a PCR-based assay (out-out PCR) used to detect targeted integration of the Clover or mRuby2 sequences at the N-terminus of Lamin A/C. Primers are located outside of the homology arms and are designed to yield a longer PCR product if the fluorescent protein is inserted. The upper band representing the targeted integration of either Clover or mRuby2 was TOPO cloned and sequenced to confirm the accurate integration of the fluorescent proteins via HDR. (b) The lower band from (a) representing non-targeted LMNA alleles was TOPO cloned, sequenced and analyzed using the web tool CrispRVariantsLite. (c) ATP1A1 alleles from the same experiment were also sequenced and non-HDR alleles were analyzed as in (b). Samples are from the experiment shown in Figure 2.
Ref: Lindsay, H. et al. CrispRVariants charts the mutation spectrum of genome engineering experiments. Nat Biotechnol. 34, 701-702 (2016)
Supplementary Figure 9 Selection for CRISPR-driven targeted integration at endogenous loci by coediting ATP1A1 via HDR.
(a) Targeting scheme for the integration of mAG1 to the C-terminus of H2BK. (b) K562 cells were transfected with a vector expressing eSpCas9(1.1) and tandem U6-driven sgRNAs targeting ATP1A1 and HIST1H2BK along with ATP1A1 ssODN RD and a plasmid donor containing a mAG1 cassette. In addition, cells were transfected with a wild-type SpCas9 expression vector and two independent sgRNA vectors along with donors. Cells were treated or not with 0.5μM ouabain for 14 days starting 3 days post-transfection and flow cytometry was used to determine the % of mAG1 positive cells in each population. (c) Fluorescence imaging of ouabain-treated cells expressing the H2BK-mAG1 fusion. Scale bar, 10μm (d) FACS-based quantification of targeted integration of a PGK1-EGFP-pA expression cassette at HPRT1 following co-selection using WT SpCas9 and various amounts of sgRNAs. (e) FACS-based quantification of targeted integration of a SA-2A-EGFP-pA gene trap cassette at AAVS1 following co-selection using an all-in-one vector expressing ATP1A1 and AAVS1 sgRNAs in addition to eSpCas9(1.1). (f) Same as in (e) but using a PGK1-TurboGFP-pA cassette. Transfection conditions are indicated in Supplementary Table 2.
Supplementary Figure 10 Endogenous tagging of the NuA4–TIP60 complex in coselected HEK293 cells.
(a) Schematic representation of the experiment. (b) Gene tagging scheme. (c) Western blot analysis on whole cell extracts from HEK293 cells to detect the expression of EPC1-tag and EP400-tag proteins. The FLAG M2 antibody was used to detect tagged proteins and the tubulin antibody was used as a loading control.
Supplementary Figure 11 Introduction of the sickle mutation in primary human cord blood (CB) CD34+ cells by coediting ATP1A1 via HDR.
(a) Typical selection process. Timing can vary according to initial modification rates at ATP1A1. (b) Schematic representation of SpCas9 target site in ATP1A1 and predicted HDR outcome dictated by the ssODN donor used to introduce the Q118R and N129D mutations. Annotated are novel restriction sites to monitor the insertion of ssODN-specified mutations. (c) Cultured CD34+ cells were electroporated with ATP1A1 and HBB RNPs along with HBB ssODN #1 and ATP1A1 G4 RD ssODN and treated as shown in (a). Genomic DNA was harvested at indicated time points and a BmgBI RFLP assay was used to determine the frequency of SpCas9-induced HDR at ATP1A1, which is indicated as the % HDR at the base of each lane. Recombinant Cas9 was used as a negative control (-). (d) Same as in (c) but using the Surveyor assay to determine the total frequency of edited alleles at ATP1A1 (NHEJ + HDR). (e) Schematic representation of SpCas9 target site in HBB and predicted HDR outcome dictated by ssODNs donor #2 used to introduce the E6V mutation. Annotated are the positions of E6 residue, 5’ UTR, protospacer adjacent motifs (PAM) and novel restriction site to monitor the insertion of ssODN-specified mutations. (f) Same as in (c) but using HBB ssODN #2 and a PstI RFLP assay to determine the frequency of SpCas9-induced HDR at HBB. (g) Same as in (f) but using the Surveyor assay to determine the total frequency of HBB edited alleles (NHEJ + HDR). (h) Same as in (f) but using a BmgBI RFLP assay to determine the frequency of SpCas9-induced HDR at ATP1A1. (i) Same as in (g) but using the Surveyor assay to determine the total frequency of ATP1A1 edited alleles (NHEJ + HDR).
Supplementary Figure 12 eSpCas9(1.1) and AsCpf1 vectors for targeting ATP1A1, available from Addgene.
Ref for pY036: Engineered Cpf1 Enzymes with Altered PAM Specificities. Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang. bioRxiv 091611; doi: https://doi.org/10.1101/091611
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Agudelo, D., Duringer, A., Bozoyan, L. et al. Marker-free coselection for CRISPR-driven genome editing in human cells. Nat Methods 14, 615–620 (2017). https://doi.org/10.1038/nmeth.4265
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DOI: https://doi.org/10.1038/nmeth.4265
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