PMID- 37590503
OWN - NLM
STAT- MEDLINE
DCOM- 20230821
LR  - 20230901
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 198
DP  - 2023 Aug 4
TI  - High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using 
      Electroporated Cas9-sgRNA Complexes.
LID - 10.3791/65264 [doi]
AB  - 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.
FAU - Craft, Julia
AU  - Craft J
AD  - Department of Internal Medicine, University of California, Davis.
FAU - Truong, Tina
AU  - Truong T
AD  - Department of Internal Medicine, University of California, Davis.
FAU - Penn, Bennett H
AU  - Penn BH
AD  - Department of Internal Medicine, University of California, Davis; Department of 
      Medical Microbiology and Immunology, University of California, Davis; 
      bhpenn@ucdavis.edu.
LA  - eng
GR  - R01 AI144149/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Video-Audio Media
DEP - 20230804
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
SB  - IM
MH  - Animals
MH  - Mice
MH  - *CRISPR-Cas Systems
MH  - *RNA, Guide, CRISPR-Cas Systems
MH  - Macrophages
MH  - Cell Line
MH  - Electroporation
EDAT- 2023/08/17 18:42
MHDA- 2023/08/21 06:43
CRDT- 2023/08/17 15:22
PHST- 2023/08/21 06:43 [medline]
PHST- 2023/08/17 18:42 [pubmed]
PHST- 2023/08/17 15:22 [entrez]
AID - 10.3791/65264 [doi]
PST - epublish
SO  - J Vis Exp. 2023 Aug 4;(198). doi: 10.3791/65264.