Department of Obstetrics and Gynecology and Columbia University Fertility Center, Columbia University, College of Physicians & Surgeons, New York, NY, 10032, USA.
Island Fertility, Stony Brook Medicine, 500 Commack Road, Suite 202, Commack, NY, 11725, USA.
J Assist Reprod Genet. 2020 Apr;37(4):735-745. doi: 10.1007/s10815-020-01723-8. Epub 2020 Mar 11.
To correct a potentially damaging mutation in haploid human embryonic stem cells.
Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP.
104,686 haploid GFP + cells were collected. Cells were cultured, individual colonies picked, and 48 clones were sent for Sanger sequencing. CRIPSR efficiency was 77.1%, with 7/48 (14.6%) clones resulting in a corrected SLC10A2 mutation. Confirmation of persistence of haploid cells was achieved with repeated FACS sorting and centromere quantification. Given the large number of passages and exposure to CRISPR, we also performed analysis of karyotypes and of off-target effects. Cells evaluated were karyotypically normal and there was no evident off target effects.
CRISPR/Cas9 can be effectively utilized to edit mutations in haploid human embryonic stem cells. Establishment and maintenance of a haploid cell culture provides a novel way to utilize CRISPR/Cas9 in gene editing, particularly in the study of recessive alleles.
纠正单倍体人类胚胎干细胞中的潜在有害突变。
对孤雌生殖衍生的胚胎干细胞系（pES12）中提取的 DNA 进行外显子组测序。选择 SLC10A2 基因突变作为本概念验证研究中感兴趣的突变，以尝试在人类多能性单倍体细胞中进行纠正。验证了突变的确认，并设计了指导 RNA 和校正模板，为 CRISPR 做准备。单倍体细胞经历了一系列荧光激活细胞分选（FACS），用 Hoechst 33342 来创建一个越来越单倍体（1n）富集的培养物。对 p.37 进行核转染，然后对 1n DNA 含量进行分选，并用 +GFP 鉴定表达 GFP 标记的 Cas9 的单倍体细胞。
共收集了 104,686 个单倍体 GFP+细胞。培养细胞，挑选单个菌落，将 48 个克隆送去进行 Sanger 测序。CRISPR 效率为 77.1%，有 7/48（14.6%）个克隆导致 SLC10A2 突变得到纠正。通过重复 FACS 分选和着丝粒定量，实现了单倍体细胞持久性的确认。考虑到大量的传代和对 CRISPR 的暴露，我们还进行了染色体组型分析和脱靶效应分析。评估的细胞染色体组型正常，没有明显的脱靶效应。
CRISPR/Cas9 可有效地用于编辑单倍体人类胚胎干细胞中的突变。建立和维持单倍体细胞培养物为基因编辑中使用 CRISPR/Cas9 提供了一种新方法，特别是在隐性等位基因的研究中。
