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CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs - PubMed

CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs

Clayton E Friedman et al. J Mol Cell Cardiol. 2023 Jun.

Abstract

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different variants. We differentiated these hiPSCs to cardiomyocytes and show MHC-β fusion proteins can localize as expected. Additionally, single-cell contractility analyses revealed cardiomyocytes with a pathogenic, hypertrophic cardiomyopathy-associated MYH7 variant exhibit salient HCM physiology relative to isogenic controls. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of functional transgenic cell lines at unprecedented scale.

Keywords: Cardiomyocytes; Disease modelling; Gene editing; Human induced pluripotent stem cells; Hypertrophic cardiomyopathy; MYH7.

Published by Elsevier Ltd.

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Conflict of interest statement

Declaration of Competing Interest C.E.M. is an equity holder in Sana Biotechnology. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Veterans Affairs or the United States Government.

Figures

Figure 1.
Figure 1.. CRaTER enriches for hiPSCs with a biallelically-edited, heterozygous variant knocked into the endogenous MYH7 locus.

(A) Schematic of gene-editing and enrichment strategy with

CR

ISPR

a

On-

T

arget

E

diting

R

etrieval (CRaTER). Human induced pluripotent stem cells (hiPSCs) stably expressing dCas9-VPR from the CLYBL locus (see Figure S1) are edited via transfection with Cas9 protein, sgRNA targeting MYH7 intron 21 and 24, and equal proportions of pJet-MYH7-eGFP-PGK-PuroR (WT MYH7) and pJet-MYH7-mTagBFP2-EF1α-BSD (E848E [GAG>GAA] MYH7) plasmid DNA repair templates (see Figure 1B and S2A). Edited cells undergo two days of antibiotic selection and then are transfected with three sgRNA targeting the MYH7 transcription start site (TSS) to transiently overexpress MYH7. Three days later, eGFP+/mTagBFP2+ hiPSCs (MYH7WT-eGFP/E848E(GAG>GAA)-mTagBFP2) are sorted by fluorescence-activated cell sorting (FACS). (B) Homology directed repair of the endogenous MYH7 locus with either plasmid DNA repair template (see Figure S2). HA= homology arm; eGFP= enhanced green fluorescent protein; pA= polyadenylation signal; PGK= mouse phosphoglycerate kinase 1 promoter; PuroR= puromycin N-acetyltransferase; mTagBFP2= enhanced monomeric blue fluorescent protein; EF1α= promoter for human elongation factor EF-1α, and BSD= blasticidin S deaminase. (C) Flow cytometry plot comparing fluorescence intensities of mTagBFP2 and eGFP in post-antibiotic selected, gene-edited hiPSCs three days after CRISPRa transfection. Sorted eGFP+/mTagBFP2+ hiPSCs shown in black box. (D) Bulk genotyping of unedited WT WTC11 hiPSCs (WT), biallelically-edited CLYBLdCas9-VPR/dCas9-VPR hiPSCs before (Edit) and after (Edit + abx selection) antibiotics selection, and biallelically edited CLYBLdCas9-VPR/dCas9-VPR hiPSCs after antibiotics selection and CRaTER enrichment (Edit + abx selection + CRaTER). The unedited (WT; 2060 bp) and knock-in (KI; 1625 bp) MYH7 products were specifically amplified from the endogenous MYH7 locus (see Figure S2A). (E) Individual colonies from one experiment of post-antibiotic selected, gene-edited hiPSCs were isolated for genotyping the MYH7 locus before (left) and after (right) CRaTER enrichment. Unedited= biallelic eGFP+/mTagBFP2+ knock-in without any variants; het. = one unedited and one knock-in allele.

Figure 2.
Figure 2.. CRaTER enriches for hiPSCs with a biallelically-edited, heterozygous variant library knocked into the endogenous MYH7 locus.

(A) Clinical significance of single, heterozygous variants within a mutation hotspot region of MYH7 exon 22 (ClinVar repository as of 1 August 2022). Variants of unknown significance (VUS), pathogenic/likely pathogenic (P/LP), and conflicting interpretations (Conflicting) indicated by grey circles, red squares, and pink triangles, respectively. (B) Homology directed repair of the endogenous MYH7 locus with either plasmid DNA repair template. Within the pJet-MYH7-mTagBFP2-EF1α-BSD repair template only, amino acid positions 848, 850, 852, 865, 866 of MYH7 exon 22 were mutagenized to saturation. See Figure S2 for metrics of the pDNA variant library and Table S1 for associated variant read counts. (C) Flow cytometry plot comparing fluorescence intensities of mTagBFP2 and eGFP in post-antibiotic selected, gene-edited hiPSCs three days after CRISPRa transfection. Sorted eGFP+/mTagBFP2+ hiPSCs shown in black box. (D) Bulk genotyping of unedited WT WTC11 hiPSCs (WT), biallelically-edited CLYBLdCas9-VPR/dCas9-VPR hiPSCs before (Edit) and after (Edit + abx selection) antibiotics selection, and biallelically edited CLYBLdCas9-VPR/dCas9-VPR hiPSCs after antibiotics selection and CRaTER enrichment (Edit + abx selection + CRaTER). The unedited (WT; 2060 bp) and knock-in (KI; 1625 bp) MYH7 products were specifically amplified from the endogenous MYH7 locus. (E) Individual colonies from one experiment of post-antibiotic selected, gene-edited hiPSCs were isolated for genotyping the MYH7 locus before (left) and after (right) CRaTER enrichment. (F) Histogram of isolated and genotyped eGFP+/mTagBFP2+ hiPSC colonies ranked by frequency in next-generation sequencing (NGS) data and binned by decile. Plot not displaying one variant that failed to pass NGS threshold and six colonies with biallelic knock-in of transgenes without any variants. See Figure S3 for metrics of the hiPSC variant library and Table S2 for associated variant read counts. (G) Position map displaying missense variants at targeted positions detected using NGS. Green= present; grey= absent. (H) Maximum number of reported variants generated in hiPSCs using serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN; 11 variants) [45], dual-integrase cassette exchange (DICE; 14 variants) [9], single-stranded oligodeoxyribonucleic (ssODN; 20 variants) [42], and CRaTER (113 variants). Note that the ssODN approach generated homozygous variants, whereas DICE and CRaTER (as used here) generates cells with heterozygous variants.

Figure 3.
Figure 3.. CRaTER enriched hiPSCs differentiate to functional cardiomyocytes and MHC-β fusion proteins localize to sarcomeres.

(A) Schematic of cardiac-directed differentiation from pluripotency to functional cardiomyocytes. (B) Spinning disk confocal micrographs (100x) of live, day 39 cardiomyocytes differentiated from MYH7WT-eGFP/WT-mTagBFP2 double knock-in (DKI) hiPSCs. MHC-β-eGFP (green) and MHC-β-mTagBFP2 (blue) expression shown in left and right images, respectively. Main scale bar= 25 μm; inset scale bar= 5 μm. See Movie S1 for video of spontaneous cardiomyocyte contraction. (C) Flow cytometry plot comparing fluorescence intensities of mTagBFP2 and eGFP in day 25 cardiomyocytes differentiated from variant library hiPSCs (from Figure 2B–F). Sorted eGFP+/mTagBFP2+ cardiomyocytes shown in black box. (D) Position map displaying missense variants at targeted amino acid positions detected using NGS of day 25 sorted cardiomyocytes (from Figure 3D). Green= present; grey= absent. See Figure S6 for metrics of the variant library and Table S3 for variant read counts in cardiomyocytes.

Figure 4.
Figure 4.. Single-cell contractility analysis of cardiomyocytes differentiated from CRaTER enriched hiPSCs.

(A) Schematic of experimental design used to measure cardiomyocyte single-cell contractility by traction force microscopy (TFM). (B) Spinning disk confocal micrographs (40x) of live, day 35 cardiomyocytes differentiated from MYH7WT-eGFP/S866S-mTagBFP2 (control; left) and MYH7WT-eGFP/M852T-mTagBFP2 (P/LP; right) hiPSCs. MHC-β-eGFP expression (green) and 0.5 μm fluorescent bead localization (red) shown in left and right images, respectively, selected from recorded videos used for TFM. Scale bar= 10 μm. See Movies S2–3 for videos of spontaneous cardiomyocyte contraction. Mean maximal forces (C), beat rates (D), and mean maximal contraction (E) and relaxation (F) velocities recorded from individual day 35 MYH7WT-eGFP/S866S-mTagBFP2 (n= 17 cells) and MYH7WT-eGFP/M852T-mTagBFP2 (n= 32 cells) cardiomyocytes. Cells imaged in (B) closely match mean forces of each genotype and are indicated as blue dots in (C-F). Bar height indicates the mean for each condition. Error bars indicate standard error of the mean. Cells measured from n= 3 biological replicates each. P values calculated using an unpaired, two-tailed t tests; p<0.05 considered significant. See Figure S8 for analysis of cell area.

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