Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility
Human germline gene correction by CRISPR/Cas9 holds great promise to eliminate transmission of genetic mutations. Recent studies have however made troublesome observations of mosaicism (the occurrence of multiple genetic events in one embryo) and loss-of-heterozygosity (LOH, loss of the allele of on...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
World Scientific Publishing
2022-09-01
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| Series: | Fertility & Reproduction |
| Online Access: | https://www.worldscientific.com/doi/10.1142/S2661318222740012 |
| _version_ | 1828190162136334336 |
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| author | Bieke BEKAERT Annekatrien BOEL Lisa DE WITTE Mina POPOVIC Panagiotis STAMATIADIS Gwenny COSEMANS Lise TORDEURS Susana MARINA CHUVA DE SOUSA LOPES Petra DE SUTTER Björn MENTEN Dominic STOOP Paul COUCKE Björn HEINDRYCKX |
| author_facet | Bieke BEKAERT Annekatrien BOEL Lisa DE WITTE Mina POPOVIC Panagiotis STAMATIADIS Gwenny COSEMANS Lise TORDEURS Susana MARINA CHUVA DE SOUSA LOPES Petra DE SUTTER Björn MENTEN Dominic STOOP Paul COUCKE Björn HEINDRYCKX |
| author_sort | Bieke BEKAERT |
| collection | DOAJ |
| description | Human germline gene correction by CRISPR/Cas9 holds great promise to eliminate transmission of genetic mutations. Recent studies have however made troublesome observations of mosaicism (the occurrence of multiple genetic events in one embryo) and loss-of-heterozygosity (LOH, loss of the allele of one of the parents), the latter being either caused by inter-homologue homologous recombination (IH-HR) or chromosome loss events. As a proof-of-concept we targeted a heterozygous base pair substitution mutation in PLCZ1, leading to subfertility. After CRISPR/Cas9 editing, we detected in 26% (7/27) of the targeted embryos that originated from mutant sperm only, wild-type alleles upon analysis, which were not obtained by exogenous repair template use. In these seven embryos, we observed a significant amount of LOH events (14%) that spanned regions beyond the PLCZ1 gene, but did not stretch the complete analysed region, pointing to IH-HR, next to a large proportion of samples (57%) showing potential but non-detectable (i.e. below the resolution of our assay) LOH. In addition, a minority of embryos (29%) showed LOH across the whole analysed region. Remarkably, also 10% of the embryos originating from wild-type sperm showed LOH events, underscoring the subpar specificity of the CRIPSR/Cas9 system. Single-cell analysis demonstrated mosaicism in 80% (4/5) of the targeted embryos, warranting caution for trophectoderm biopsy interpretation in future human embryo editing attempts. Lastly, a correction rate of 75% was observed in our single-cell data which illustrates that the correction rate was possibly underestimated when looking at the whole-embryo level. |
| first_indexed | 2024-04-12T08:18:04Z |
| format | Article |
| id | doaj.art-bf1c12c678654bb397d64a33a8373934 |
| institution | Directory Open Access Journal |
| issn | 2661-3182 2661-3174 |
| language | English |
| last_indexed | 2024-04-12T08:18:04Z |
| publishDate | 2022-09-01 |
| publisher | World Scientific Publishing |
| record_format | Article |
| series | Fertility & Reproduction |
| spelling | doaj.art-bf1c12c678654bb397d64a33a83739342022-12-22T03:40:44ZengWorld Scientific PublishingFertility & Reproduction2661-31822661-31742022-09-010403n04898910.1142/S2661318222740012Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary InfertilityBieke BEKAERT0Annekatrien BOEL1Lisa DE WITTE2Mina POPOVIC3Panagiotis STAMATIADIS4Gwenny COSEMANS5Lise TORDEURS6Susana MARINA CHUVA DE SOUSA LOPES7Petra DE SUTTER8Björn MENTEN9Dominic STOOP10Paul COUCKE11Björn HEINDRYCKX12Ghent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumCenter for Medical Genetics Ghent from the Department of Biomolecular Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumCenter for Medical Genetics Ghent from the Department of Biomolecular Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumCenter for Medical Genetics Ghent from the Department of Biomolecular Medicine, Ghent, BelgiumGhent-Fertility and Stem Cell Team (G-FaST) from the Department for Reproductive Medicine, Ghent, BelgiumHuman germline gene correction by CRISPR/Cas9 holds great promise to eliminate transmission of genetic mutations. Recent studies have however made troublesome observations of mosaicism (the occurrence of multiple genetic events in one embryo) and loss-of-heterozygosity (LOH, loss of the allele of one of the parents), the latter being either caused by inter-homologue homologous recombination (IH-HR) or chromosome loss events. As a proof-of-concept we targeted a heterozygous base pair substitution mutation in PLCZ1, leading to subfertility. After CRISPR/Cas9 editing, we detected in 26% (7/27) of the targeted embryos that originated from mutant sperm only, wild-type alleles upon analysis, which were not obtained by exogenous repair template use. In these seven embryos, we observed a significant amount of LOH events (14%) that spanned regions beyond the PLCZ1 gene, but did not stretch the complete analysed region, pointing to IH-HR, next to a large proportion of samples (57%) showing potential but non-detectable (i.e. below the resolution of our assay) LOH. In addition, a minority of embryos (29%) showed LOH across the whole analysed region. Remarkably, also 10% of the embryos originating from wild-type sperm showed LOH events, underscoring the subpar specificity of the CRIPSR/Cas9 system. Single-cell analysis demonstrated mosaicism in 80% (4/5) of the targeted embryos, warranting caution for trophectoderm biopsy interpretation in future human embryo editing attempts. Lastly, a correction rate of 75% was observed in our single-cell data which illustrates that the correction rate was possibly underestimated when looking at the whole-embryo level.https://www.worldscientific.com/doi/10.1142/S2661318222740012 |
| spellingShingle | Bieke BEKAERT Annekatrien BOEL Lisa DE WITTE Mina POPOVIC Panagiotis STAMATIADIS Gwenny COSEMANS Lise TORDEURS Susana MARINA CHUVA DE SOUSA LOPES Petra DE SUTTER Björn MENTEN Dominic STOOP Paul COUCKE Björn HEINDRYCKX Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility Fertility & Reproduction |
| title | Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility |
| title_full | Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility |
| title_fullStr | Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility |
| title_full_unstemmed | Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility |
| title_short | Correcting a PLC Mutation in the Human Germ Line to Overcome Hereditary Infertility |
| title_sort | correcting a plc mutation in the human germ line to overcome hereditary infertility |
| url | https://www.worldscientific.com/doi/10.1142/S2661318222740012 |
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