Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls

Dina Simkin; Vasileios Papakis; Bernabe I. Bustos; Christina M. Ambrosi; Steven J. Ryan; Valeriya Baru; Luis A. Williams; Graham T. Dempsey; Owen B. McManus; John E. Landers; Steven J. Lubbe; Alfred L. George; Evangelos Kiskinis

doi:10.1016/j.stemcr.2022.02.008

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls

Dina Simkin, Vasileios Papakis, Bernabe I. Bustos, Christina M. Ambrosi, Steven J. Ryan, Valeriya Baru, Luis A. Williams, Graham T. Dempsey, Owen B. McManus, John E. Landers, Steven J. Lubbe, Alfred L. George, Evangelos Kiskinis^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

33 Scopus citations

Abstract

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

Original language	English (US)
Pages (from-to)	993-1008
Number of pages	16
Journal	Stem cell reports
Volume	17
Issue number	4
DOIs	https://doi.org/10.1016/j.stemcr.2022.02.008
State	Published - Apr 12 2022

Funding

We are grateful to the following funding sources: US National Institutes of Health ( NIH ) National Institute on Neurological Disorders and Stroke (NINDS) U54NS108874 (A.L.G. and E.K.), NIH /NINDS and National Institute on Aging (NIA) R01NS104219 (E.K.), NIH /NINDS R01NS073873 (J.E.L.) and R56NS073873 (J.E.L.), the Les Turner ALS Foundation (E.K.), and the New York Stem Cell Foundation (E.K.). We are grateful to Bill Skarnes and Justin McDonough for generation of the NEK1-02 iPSC isogenic line. E.K. is a Les Turner ALS Center Investigator and a New York Stem Cell Foundation – Robertson Investigator.

Keywords

CRISPR/Cas9
KCNQ2
WGS
disease modeling
genome editing
iPSCs
isogenic control lines
mtDNA
on-target insertions/deletions

ASJC Scopus subject areas

Genetics
Biochemistry
Cell Biology
Developmental Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1016/j.stemcr.2022.02.008

Cite this

Simkin, D., Papakis, V., Bustos, B. I., Ambrosi, C. M., Ryan, S. J., Baru, V., Williams, L. A., Dempsey, G. T., McManus, O. B., Landers, J. E., Lubbe, S. J., George, A. L., & Kiskinis, E. (2022). Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls. Stem cell reports, 17(4), 993-1008. https://doi.org/10.1016/j.stemcr.2022.02.008

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title = "Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls",

abstract = "The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.",

keywords = "CRISPR/Cas9, KCNQ2, WGS, disease modeling, genome editing, iPSCs, isogenic control lines, mtDNA, on-target insertions/deletions",

author = "Dina Simkin and Vasileios Papakis and Bustos, {Bernabe I.} and Ambrosi, {Christina M.} and Ryan, {Steven J.} and Valeriya Baru and Williams, {Luis A.} and Dempsey, {Graham T.} and McManus, {Owen B.} and Landers, {John E.} and Lubbe, {Steven J.} and George, {Alfred L.} and Evangelos Kiskinis",

note = "Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2022",

month = apr,

day = "12",

doi = "10.1016/j.stemcr.2022.02.008",

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Simkin, D, Papakis, V, Bustos, BI, Ambrosi, CM, Ryan, SJ, Baru, V, Williams, LA, Dempsey, GT, McManus, OB, Landers, JE, Lubbe, SJ, George, AL & Kiskinis, E 2022, 'Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls', Stem cell reports, vol. 17, no. 4, pp. 993-1008. https://doi.org/10.1016/j.stemcr.2022.02.008

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T1 - Homozygous might be hemizygous

T2 - CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls

AU - Simkin, Dina

AU - Papakis, Vasileios

AU - Bustos, Bernabe I.

AU - Ambrosi, Christina M.

AU - Ryan, Steven J.

AU - Baru, Valeriya

AU - Williams, Luis A.

AU - Dempsey, Graham T.

AU - McManus, Owen B.

AU - Landers, John E.

AU - Lubbe, Steven J.

AU - George, Alfred L.

AU - Kiskinis, Evangelos

PY - 2022/4/12

Y1 - 2022/4/12

N2 - The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

AB - The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

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KW - on-target insertions/deletions

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Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls

Abstract

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

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