利用 CRISPR 基因编辑技术生成人类同源诱导多能干细胞系。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY
Lori L Bonnycastle, Amy J Swift, Erin C Mansell, Angela Lee, Elizabeth Winnicki, Elizabeth S Li, Catherine C Robertson, Victoria A Parsons, Trung Huynh, Chad Krilow, Karen L Mohlke, Michael R Erdos, Narisu Narisu, Francis S Collins
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引用次数: 0

摘要

我们开发了一种高效的 CRISPR 基因编辑方案,并生成了携带六个 2 型糖尿病基因位点(ABCC8、MTNR1B、TCF7L2、HNF4A、CAMK1D 和 GCK)假定致病单核苷酸变异杂合或同源等位基因的同源诱导多能干细胞 (iPSC) 株系。我们采用了基于序列的两步法,首先识别出具有最高编辑细胞比例的转染细胞池,这大大减少了下游分离单个编辑细胞克隆的工作。我们发现,素体编辑能对 iPSC 进行有针对性的基因改变,而优化系统组件和引导 RNA 设计对达到可接受的效率至关重要。利用 PEmax、epgRNA 修饰和 MLH1dn 的系统带来了显著的益处,编辑效率达到 36-73%。每个变体所需的编辑成功率和 pegRNA 设计优化因目标位点的序列而异。只要注意设计,质粒编辑是生成同源 iPSC 株系的一种很有前景的方法,可在共同的遗传背景下研究特定的基因变化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing.

We developed an efficient CRISPR prime editing protocol and generated isogenic-induced pluripotent stem cell (iPSC) lines carrying heterozygous or homozygous alleles for putatively causal single nucleotide variants at six type 2 diabetes loci (ABCC8, MTNR1B, TCF7L2, HNF4A, CAMK1D, and GCK). Our two-step sequence-based approach to first identify transfected cell pools with the highest fraction of edited cells significantly reduced the downstream efforts to isolate single clones of edited cells. We found that prime editing can make targeted genetic changes in iPSC and optimization of system components and guide RNA designs that were critical to achieve acceptable efficiency. Systems utilizing PEmax, epegRNA modifications, and MLH1dn provided significant benefit, producing editing efficiencies of 36-73%. Editing success and pegRNA design optimization required for each variant differed depending on the sequence at the target site. With attention to design, prime editing is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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