Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells Journal Article
| Authors: | Wu, Y.; Sidharta, M.; Zhong, A.; Persily, B.; Li, M.; Zhou, T. |
| Article Title: | Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells |
| Abstract: | Prime editing introduces single-nucleotide polymorphism changes, small deletions, or insertions at a specific genome site without double-stranded DNA breaks or the need for the donor template. Here, we present a protocol to design, conduct, and evaluate prime editing in human pluripotent stem cells. We describe steps for pegRNA and nicking sgRNA design and cloning, the prime editing tool electroporation, and the efficiency evaluation using Miseq. We elaborate the process of GBA (N370S) mutation induction and correction as an example. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1 © 2023 The Authors |
| Keywords: | controlled study; gene mutation; human cell; single nucleotide polymorphism; gene deletion; polymerase chain reaction; amplicon; escherichia coli; stem cells; double stranded dna break; electroporation; pluripotent stem cell; gene insertion; complementary dna; cell cloning; ampicillin; endotoxin; sanger sequencing; human; article; crispr; clustered regularly interspaced short palindromic repeat; gene editing; crispr-cas9 system; carbenicillin; enzyme assisted extraction |
| Journal Title: | STAR Protocols |
| Volume: | 4 |
| Issue: | 4 |
| ISSN: | 2666-1667 |
| Publisher: | Cell Press |
| Date Published: | 2023-12-15 |
| Start Page: | 102583 |
| Language: | English |
| DOI: | 10.1016/j.xpro.2023.102583 |
| PROVIDER: | scopus |
| PMCID: | PMC10519846 |
| PUBMED: | 37738119 |
| DOI/URL: | |
| Notes: | The MSK Cancer Center Support Grant (P30 CA008748) is acknowledged in the PDF. Corresponding MSK author is Youjun Wu-- Source: Scopus |
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