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Curr Protoc Mol Biol. 2017 Oct 2;120:31.10.1-31.10.19. doi: 10.1002/cpmb.43.

Production of Purified CasRNPs for Efficacious Genome Editing.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, California.
2
Innovative Genomics Institute, University of California, Berkeley, California.
3
QB3 MacroLab, University of California, Berkeley, California.

Abstract

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells.

KEYWORDS:

CRISPR; Cas9; RNP; genome editing; ribonucleoprotein

PMID:
28967993
DOI:
10.1002/cpmb.43
[Indexed for MEDLINE]

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