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

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

doi:10.1016/j.stemcr.2022.02.008

Cited by 40 publications

(46 citation statements)

References 75 publications

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“…Bi-allelic amplification can be confirmed in heterozygous clones, but not in the case of homozygous clones, which lack SNPs or other genomic events, evidencing the amplification of both alleles [ 44 ]. Thereby, all homozygous clones were subjected to PCR amplification of the largest region on the PDE6C locus.…”

Section: Resultsmentioning

confidence: 99%

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High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs

Siles

Gaudó

Pomares

2023

IJMS

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Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies thatcurrently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.

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Section: Resultsmentioning

confidence: 99%

“…On-target anomalies and off-targets are two of the main problems of the CRISPR/Cas9 system [ 41 , 44 , 45 , 57 ]. Strikingly, on-target effects have been reported in the literature in up to 40% of hiPSC clones after CRISPR editing [ 57 ].…”

Section: Discussionmentioning

confidence: 99%

High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs

Siles

Gaudó

Pomares

2023

IJMS

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“…Therefore, the lines generated for this work were validated to be free of unwanted mutations by first probing for fragments of the donor plasmid ( Figures S1 E and S1F). In addition, quantitative genotyping PCR (qgPCR) was also carried out to confirm that the lines were indeed homozygous and not false positives ( Figure S1 G) ( Simkin et al., 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Characterizing arrhythmia using machine learning analysis of Ca2+ cycling in human cardiomyocytes

et al. 2022

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“…However interpretation of such data must be done with caution, donor chromosome sequences (including the drive element) may incorrectly appear homozygous when NHEJ mutations cause the binding sites of a PCR primer to be blocked on the recipient chromosome. This issue has been raised in several analyses 42 , 54 – 56 and highlights potential pitfalls for identifying homing events with these types of molecular assays. We highlight these cases specifically because we believe such genetic assays are worth perusing, but should be informed by this prior work to reduce the chance of misinterpretation.…”

Section: Discussionmentioning

confidence: 99%

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias

Verkuijl

González

et al. 2022

Nat Commun

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CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

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

Cited by 40 publications

References 75 publications

High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs

High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs

Characterizing arrhythmia using machine learning analysis of Ca2+ cycling in human cardiomyocytes

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias

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