Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells

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....

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Detalles Bibliográficos
Autores principales: Wu, Youjun, Sidharta, Mega, Zhong, Aaron, Persily, Benjamin, Li, Mu, Zhou, Ting
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10519846/
https://www.ncbi.nlm.nih.gov/pubmed/37738119
http://dx.doi.org/10.1016/j.xpro.2023.102583
Descripción
Sumario: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)

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