CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells.
Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.
Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY, 10065, USA.|Weill Graduate School of Medical Sciences at Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional M.D.-Ph.D. Program, 1300 York Avenue, New York, NY, 10065, USA.
Nipun
N
Verma
Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY, 10065, USA.
Zengrong
Z
Zhu
Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY, 10065, USA. huangfud@mskcc.org.
Danwei
D
Huangfu
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004318:Doxycycline / Q000494:pharmacology; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D001426:Bacterial Proteins; D000939:Epitopes; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D004318:Doxycycline
CRISPR/Cas; Epitope tag; Fluorescent reporter; Gene targeting; Homologous recombination; Human pluripotent stem cells (hPSCs); Knockin