Limitations of gene editing assessments in human preimplantation embryos.

Autor: Liang D; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA.; Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei, 230022, Anhui, China., Mikhalchenko A; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Ma H; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Marti Gutierrez N; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Chen T; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China., Lee Y; Department of Biomedical Science, College of Life Science and CHA Advanced Research Institute, CHA University, Seongnam, Gyeonggi, 13488, Republic of Korea.; Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505, Republic of Korea., Park SW; Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea.; School of Dentistry, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, Republic of Korea., Tippner-Hedges R; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Koski A; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Darby H; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Li Y; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Van Dyken C; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., Zhao H; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China., Wu K; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China., Zhang J; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China., Hou Z; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China., So S; Department of Biomedical Science, College of Life Science and CHA Advanced Research Institute, CHA University, Seongnam, Gyeonggi, 13488, Republic of Korea., Han J; Department of Biomedical Science, College of Life Science and CHA Advanced Research Institute, CHA University, Seongnam, Gyeonggi, 13488, Republic of Korea., Park J; Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505, Republic of Korea., Kim CJ; Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505, Republic of Korea., Zong K; Technology Center of Hefei Customs, Hefei, 230009, Anhui, China., Gong J; Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China.; China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China., Yuan Y; Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China.; China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China., Gu Y; Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China.; China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China., Shen Y; Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China.; China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China., Olson SB; Department of Molecular and Medical Genetics and Knight Diagnostic Laboratories, Oregon Health & Science University, Portland, OR, 97239, USA., Yang H; Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China., Battaglia D; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA., O'Leary T; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, OR, 97239, USA., Krieg SA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, OR, 97239, USA., Lee DM; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, OR, 97239, USA., Wu DH; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, OR, 97239, USA., Duell PB; Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, OR, 97239, USA., Kaul S; Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, OR, 97239, USA., Kim JS; Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea.; Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Republic of Korea., Heitner SB; Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, OR, 97239, USA., Kang E; Department of Biomedical Science, College of Life Science and CHA Advanced Research Institute, CHA University, Seongnam, Gyeonggi, 13488, Republic of Korea.; Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505, Republic of Korea., Chen ZJ; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA.; Center for Reproductive Medicine, Shandong University, Jinan, Shandong, 250001, China.; Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, 200127, China., Amato P; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA.; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, OR, 97239, USA., Mitalipov S; Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S. Bond Avenue, Portland, OR, 97239, USA. shoukhrat@gmail.com.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2023 Mar 07; Vol. 14 (1), pp. 1219. Date of Electronic Publication: 2023 Mar 07.
DOI: 10.1038/s41467-023-36820-6
Abstrakt: Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.
(© 2023. The Author(s).)
Databáze: MEDLINE
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