A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system

STAR Protoc. 2022 Mar 4;3(1):101216. doi: 10.1016/j.xpro.2022.101216. eCollection 2022 Mar 18.

Abstract

Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.

Keywords: Biotechnology and bioengineering; CRISPR; Cell Biology; Cell culture; Cell isolation; Molecular Biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Gene Editing / methods
  • Gene Knockout Techniques
  • Humans
  • Peroxiredoxins / genetics
  • RNA, Guide, CRISPR-Cas Systems* / genetics
  • Transfection

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • PRDX1 protein, human
  • Peroxiredoxins