High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes.
J Vis Exp
; (198)2023 08 04.
Article
en En
| MEDLINE
| ID: mdl-37590503
ABSTRACT
Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Sistemas CRISPR-Cas
/
ARN Guía de Sistemas CRISPR-Cas
Límite:
Animals
Idioma:
En
Revista:
J Vis Exp
Año:
2023
Tipo del documento:
Article