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

Friedman, Clayton E.; Fayer, Shawn; Pendyala, Sriram; Chien, Wei-Ming; Tran, Linda; Chao, Leslie; McKinstry, Ashley; Karbassi, Elaheh; Fenix, Aidan M.; Loiben, Alexander; Murry, Charles E.; Fowler, Douglas M.; Yang, Kai-Chun

doi:10.1101/2023.01.25.525582

Cited by 3 publications

(30 citation statements)

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“…To test this hypothesis, we used a previously generated, biallelically edited isogenic hiPSC library containing 113 heterozygous MYH7 missense variants encoding 78 different amino acid substitutions. 21 Within each of these diploid cells (generated on a healthy WTC11 background), there is one WT MYH7 allele C-terminally tagged with eGFP, while the other MYH7 allele contains a single MYH7 variant similarly tagged with mTagBFP2. Tagging both MYH7 alleles with different fluorescent proteins was originally designed to enable enrichment of heterozygously gene-edited cells using CRaTER.…”

Section: Resultsmentioning

confidence: 99%

“…To enable DMS in hiPSC derivatives, we leveraged a method we previously developed called CRISPRa On-Target Editing Retrieval (CRaTER) that allowed for the pooled generation of a variant library in hiPSCs for the first time. 21 This library of isogenic gene-edited hiPSCs targeted the endogenous MYH7 ( myosin heavy chain 7 ) locus with a single, heterozygous missense variant per cell to mutagenize 5 amino acid positions of MYH7 to near-saturation, generating 113 unique MYH7 missense variants. 21 MYH7 encodes a sarcomeric thick filament protein (β-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes that interacts with actin to generate contractile force.…”

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confidence: 99%

“…21 This library of isogenic gene-edited hiPSCs targeted the endogenous MYH7 ( myosin heavy chain 7 ) locus with a single, heterozygous missense variant per cell to mutagenize 5 amino acid positions of MYH7 to near-saturation, generating 113 unique MYH7 missense variants. 21 MYH7 encodes a sarcomeric thick filament protein (β-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes that interacts with actin to generate contractile force. Pathogenic autosomal-dominant missense variants in MYH7 are a leading cause of hypertrophic cardiomyopathy (HCM), 22,23 a disease characterized by pathologically thickened myocardial walls that can present as sudden cardiac death or heart failure.…”

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confidence: 99%

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Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes

Friedman,

Fayer,

Pendyala

et al. 2024

Circ: Genomic and Precision Medicine

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BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encode the sarcomeric protein (β-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of the MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that β-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of β-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of β-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance detected in patients with cardiomyopathies and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes

Friedman,

Fayer,

Pendyala

et al. 2024

Circ: Genomic and Precision Medicine

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“…One solution is to use a second selectable reporter driven by a dedicated reporter for the positive selection, which can then be removed (i.e., through Cre‐loxP recombination of CRISPR/Cas9 excision; [172]). An alternative approach is to transiently activate the silent locus through CRISPRa, so as to enable a well‐timed positive selection [173].…”

Section: Methods To Induce Gene Change‐of‐functionmentioning

confidence: 99%

“…Furthermore, heterochromatin can suppress the activity of constitutive promoters, and decrease the efficiency of HDR. CRISPRa On‐Target Editing Retrieval (CRaTER) overcomes these issues by transiently activating the target locus with CRISPRa, so as to allow the selection of cells that undergo HR of a transgene‐fluorescent protein fusion using FACS [173]. The approach was demonstrated to enable saturation mutagenesis of five amino acid residues within a mutational hotspot of MYH7 , a cardiomyopathy‐associated gene, which yielded ~ 80% of possible missense variants in hPSCs, 19 of which were clonally genotyped for further characterization in hPSC‐CMs.…”

Section: High‐throughput Functional Genomicsmentioning

confidence: 99%

Manipulating and studying gene function in human pluripotent stem cell models

Balmas,

Sozza,

Bottini

et al. 2023

FEBS Letters

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Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.

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Multiplexed functional assessments ofMYH7variants in human cardiomyocytes at scale

Friedman

Fayer

Pendyala

et al. 2023

Preprint

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Background: Single, autosomal-dominant missense mutations inMYH7, which encodes a sarcomeric protein (MHC-β) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, ~75% ofMYH7variants are of unknown significance (VUS), causing diagnostic challenges for clinicians and emotional distress for patients. Deep mutational scans (DMS) can determine variant effect at scale, but have only been utilized in easily-editable cell lines. While human induced pluripotent stem cells (hiPSCs) can be differentiated to numerous cell types that enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates. However, CRaTER enrichment has recently enabled the pooled generation of a saturated five positionMYH7variant hiPSC library suitable for DMS for the first time. Results: As a proof-of-concept, we differentiated thisMYH7variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-β variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-β protein loss occurs in a failing human heart with a pathogenicMYH7mutation. We find the multiplexed assessment of MHC-β abundance and hiPSC-CM survival both accurately segregate all pathogenic variants from synonymous controls. Overall, functional scores of 68 amino acid substitutions across these independent assays are ~50% consistent. Conclusions: This study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments ofMYH7missense variants at scale for the first time. This proof-of-concept demonstrates the ability to DMS previously restricted, clinically-actionable genes to reduce the burden of VUS on patients and clinicians.

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

Cited by 3 publications

References 44 publications

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes

Manipulating and studying gene function in human pluripotent stem cell models

Multiplexed functional assessments ofMYH7variants in human cardiomyocytes at scale

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