Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9

  • Zijun Huo
  • Jian Tu
  • Dung-Fang Lee
  • Ruiying ZhaoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2108)


CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system has been widely accepted and employed in gene editing of a vast range of cell types. Comparing to zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), CRISPR shows less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene, but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome-wide screening. Here we will describe the procedure of generating stable cell lines with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.

Key words

CRISPR Genome editing Precision gene editing gRNA design Donor vector Stable cell line validation Southern blot 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Zijun Huo
    • 1
    • 2
  • Jian Tu
    • 1
    • 3
  • Dung-Fang Lee
    • 1
  • Ruiying Zhao
    • 1
    Email author
  1. 1.Department of Integrative Biology and Pharmacology, McGovern Medical SchoolThe University of Texas Health Science Center at HoustonHoustonUSA
  2. 2.Department of EndocrinologyThe First Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China
  3. 3.Department of Musculoskeletal OncologyThe First Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China

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