Genome Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Thomson, Science 282:1145-1147, 1998; Takahashi et al. Cell 131:861-872, 2007), collectively referred to as pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement our insight gained from model organisms (Soldner et al. Cell 146:318-331, 2011; Soldner and Jaenisch, Science 338:1155-1156, 2012). Recently, genetic engineering using site-specific nucleases has been established in hPSCs (Hockemeyer et al. Nat Biotechnol 27:851-857, 2009; Hockemeyer et al., Nat Biotechnol 29:731-734, 2011; Zou et al., Cell Stem Cell 5:97-110, 2011; Yusa et al., Nature 478:391-394, 2011; DeKelver et al., Genome Res 20:1133-1142, 2010), allowing a level of genetic control previously limited to model systems. Thus, we can now perform targeted gene knockouts, generate tissue-specific cell lineage reporters, overexpress genes from a defined locus, and introduce and repair single point mutations in hPSCs. This ability to genetically engineer pluripotent stem cells will significantly facilitate the study of human disease in a defined genetic context. Here we outline protocols for efficient gene targeting in hPSCs.

Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies

“…To determine whether this finding is general, we investigated the consequence of TCAB1 or Coilin KO in the human embryonic stem cell (hESC) line WIBR#3 (Chiba and Hockemeyer, 2015). Clonal hESC lines with TCAB1 or Coilin KO were generated using the same approach that was successful in HCT116 cells (Figure 5—figure supplement 2A).…”

“…The 293T, VA-13, and U2OS cell lines are long-term Collins lab stocks. The hESC line WIBR#3 (NIH stem cell registry #0079, originating at the Whitehead Institute for Biomedical Research) was maintained on inactivated mouse embryonic fibroblasts in DMEM/F12 under conditions previously described (Chiba and Hockemeyer, 2015). None of the cell lines have been authenticated recently at the genome sequence level, but all have the expected cell morphology and doubling time.…”

Induced Pluripotent Stem Cells Meet Genome Editing

“…Some important adaptations and improvements to increase the ease and scope of Cas9-mediated genome engineering in hPSCs was the establishment of CRISPR/CAS-systems from different organisms (Hou et al, 2013; Zetsche et al, 2015) that respond to different PAM sequences or by engineering spCas9 to associate with alternative PAMs by structure-based engineering of Cas9 and thereby extending genomic target range and specificity of spCas9 (Kleinstiver et al, 2015a; Kleinstiver et al, 2015b). Furthermore, several detailed protocols that describe the implementation of genome editing techniques in pluripotent stem cell systems have been optimized and published (Blair et al, 2016; Byrne and Church, 2015; Chiba and Hockemeyer, 2015; Yusa, 2013). …”

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

“…Additionally, the use of patient-derived induced pluripotent stem cells allows the repair or introduction of known diseasecausing mutations in a patient-specific genetic background 29 , providing a platform from which to investigate disease mechanisms and test therapeutics using a patient's own cells 30 . In summary, gene editing in hPSCs is an efficient and versatile approach for investigating the basic biology of human development and disease 31 .…”