DOI: 10.24075/brsmu.2018.052

ORIGINAL RESEARCH

The efficacy of CRISPR-Cas9-mediated induction of the CCR5delta32 mutation in the human embryo

Kodyleva TA1, Kirillova AO1, Tyschik EA1, Makarov VV2, Khromov AV2, Guschin VA2, Abubakirov AN1, Rebrikov DV1,3, Sukhikh GT1
About authors

1 Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow

2 Lomonosov Moscow State University, Moscow, Russia

3 Pirogov Russian National Research Medical University, Moscow

Correspondence should be addressed: Denis V. Rebrikov
Ostrovityanova 1, Moscow, 117997; moc.liamg@vokirberd

Received: 2018-09-26 Accepted: 2018-10-09
|

The editing of the CCR5 gene in the CD4+ T cell genome is an effective way of preventing HIV-1 proliferation. Very similar strategies can be used to protect the fetus of an HIV-infected female showing a weak response to antiretroviral therapy. Inducing the “natural” CCR5delta32 mutation in a zygote may guard the fetus against HIV infection both in utero and at birth. In this study, we optimize the CRISPR-Cas9 system to induce a homozygous 32-nt deletion similar to the naturally occurring CCR5delta32 allele in the human zygote at the S-phase. Edits were done in the abnormal tripronuclear zygotes unsuitable for IVF. Sixteen tripronuclear zygotes in the S-phase obtained from WT CCR5 donors were injected with an original CRISPR-Cas9 system designed by the authors. Upon injection, the zygotes were transferred into the Blastocyst (COOK) embryo culture medium and cultured for 5 days in a CO2 incubator until blastocysts were formed (approximately 250 cells). Eight zygotes that successfully developed into blastocysts were PCR-genotyped to analyze the efficacy of genome editing. Of 16 zygotes injected with CRISPR-Cas9, only 8 reached the blastocyst stage. PCR genotyping revealed the absence of the initial WT CCR5 variant in 5 of 8 blastocysts (100% CCR5delta32 homozygous). Two had about 3% and one about 20% of WT CCR5 mosaicism. This leads us to conclude that the efficacy of the proposed CRISPR-Cas9 system for the induction of the CCR5delta32 mutation in human embryos is very high producing more than 50% of completely modified embryos.

Keywords: genome editing, CRISPR-Cas9, human embryo, CCR5, CCR5delta32, HIV resistance

КОММЕНТАРИИ (0)