Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.
Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments.
Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; Department of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
Taichi
T
Matsunaga
Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; Department of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. Electronic address: juny@frontier.kyoto-u.ac.jp.
Jun K
JK
Yamashita
D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D055786:Gene Knockout Techniques / Q000379:methods*; D015151:Immunoblotting; D051379:Mice; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
D064130:CRISPR-Associated Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D013752:Tetracycline; D004318:Doxycycline
CRISPR/Cas9 system; Endothelial cell differentiation; Knockout-rescue system; Promoter; VEGFR2/Flk1