Header

UZH-Logo

Maintenance Infos

Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells


Kissling, Lucas; Monfort, Asun; Swarts, Daan C; Wutz, Anton; Jinek, Martin (2019). Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells. In: Pyle, A M; Christianson, D W. Methods in Enzymology. Cambridge MA: Elsevier, 241-263.

Abstract

CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.

Abstract

CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.

Statistics

Citations

Dimensions.ai Metrics
4 citations in Web of Science®
4 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

0 downloads since deposited on 06 Sep 2019
0 downloads since 12 months

Additional indexing

Item Type:Book Section, refereed, further contribution
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Language:English
Date:2019
Deposited On:06 Sep 2019 08:14
Last Modified:29 Jul 2020 11:15
Publisher:Elsevier
Series Name:Methods in Enzymology
ISSN:0076-6879
OA Status:Closed
Publisher DOI:https://doi.org/10.1016/bs.mie.2018.10.028
PubMed ID:30691645

Download

Closed Access: Download allowed only for UZH members