Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes

Cai M Roberts; Elena S Ratner

doi:10.1007/978-1-0716-1162-3_18

Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes

Methods Mol Biol. 2021:2255:213-232. doi: 10.1007/978-1-0716-1162-3_18.

Authors

Cai M Roberts¹, Elena S Ratner²

Affiliations

¹ Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA. cai.roberts@yale.edu.
² Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.

PMID: 34033106
DOI: 10.1007/978-1-0716-1162-3_18

Abstract

CRISPR/Cas9-based gene editing is a recent advance that allows for the knockout or alteration of target genes within mammalian cells. Many variations of the technique exist, but here we describe two systems of plasmid-based CRISPR gene knockout which together allow for the selective knockout of virtually any gene target. Compared with other CRISPR-based systems, these plasmids have the advantages of delivering all the necessary components in one plasmid, choice of multiple selectable markers, and choice of route of administration into target cells. In addition, potential off-target effects from one system (dependent upon selection of target gene) can be overcome through use of the second system. Strategies for optimizing the knockout process and selection of finished cell lines are also presented.

Keywords: CRISPR; Cas9; Knockout; Nickase; Plasmids; Selection; Transfection; sgRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism*
CRISPR-Cas Systems*
Cells, Cultured
Gene Editing*
Gene Knockout Techniques*
Humans
Plasmids / genetics*

Substances

CRISPR-Associated Protein 9