title: TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.
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.
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.
authors.forename: William T
authors.initials: WT
authors.lastname: Hendriks
authors.affiliation: Harvard University, Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Cambridge, Massachusetts.
authors.forename: Xin
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Harvard University, Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Cambridge, Massachusetts.
authors.forename: Laurence
authors.initials: L
authors.lastname: Daheron
authors.affiliation: Harvard University, Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Cambridge, Massachusetts.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Cowan
mesh_terms: D064113:CRISPR-Cas Systems; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D003851:Deoxyribonucleases
keywords: CRISPR/Cas9; TALEN; gene editing; pluripotent stem cells; transfection