UNIT 5B.3 TALEN‐ and CRISPR/Cas9‐Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid‐Based Transfection. (3rd August 2015)

• Record Type: Journal Article
• Authors: Hendriks, William T.; Jiang, Xin; Daheron, Laurence; Cowan, Chad A.
• Is Part Of: Current protocols in stem cell biology. Volume 34(2015)
• Page Start: 5B.3.1
• Page End: 5B.3.25
• Publication Date: 2015-08-03
• Subjects: TALEN -- CRISPR/Cas9 -- gene editing -- pluripotent stem cells -- transfection; Stem cells -- Laboratory manuals; Stem cells -- Physiology; Stem Cells; Stem cells; Laboratory Manual; Laboratory manuals; 571.5
• DOI: 10.1002/9780470151808.sc05b03s34
• Languages: English
• ISSNs: 1941-7322

Abstract

Abstract : Using custom‐engineered nuclease‐mediated genome editing, such as Transcription Activator–Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA‐guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single‐stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications. © 2015 by John Wiley & Sons, Inc.