br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Human embryonic stem cells (hESCs) are capable of unlimited self-renewal in culture while maintaining the potential to differentiate into any cell type present in the human body and thus provide researchers great opportunities for human developmental studies, disease modeling, and cell-replacement therapies (Zhu and Huangfu, 2013). All these applications benefit from lineage-specific knockin reporters that allow real-time observation of gene-expression dynamics, cell-lineage tracing, and isolation of a specific cell population of interest from a heterogeneous differentiation culture for downstream analysis. However, creating knockin MC 1568 in hESCs is usually a lengthy and technically challenging process. Because of the low efficiency of homologous recombination, the donor vector needs to contain a drug-resistance gene for enrichment of cells with the correct integration. Due to the concern that the insertion of a drug-resistance cassette may interfere with the expression of the reporter gene or neighboring genes, it is usually necessary to remove the drug-resistance cassette through a second electroporation step followed by isolation of clonal lines and further characterization (Davis et al., 2008). Thus, substantial time and effort is needed to generate a knockin reporter hESC line through this two-step procedure. The development of engineered “genomic scissors” that introduce site-specific DNA double-strand breaks (DSBs), including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, has greatly facilitated gene targeting in hESCs (Kim and Kim, 2014). Repair of a DSB by non-homologous end joining (NHEJ) often results in insertion and/or deletions (Indels) that can be used to knock out a target gene in hESCs (Ding et al., 2013a, b; González et al., 2014). Alternatively, homology-directed repair (HDR) can be employed to efficiently incorporate exogenous sequences such as a fluorescent reporter into a specific genomic locus in hESCs (Hockemeyer et al., 2009, 2011; Hou et al., 2013; Merkert et al., 2014). Despite the significant improvement, a drug-resistance cassette is still required for generating knockin reporters of genes that are not expressed in undifferentiated hESCs. To overcome these limitations, we made use of the CRISPR/Cas system, in which the CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) duplex or a single chimeric guide RNA (gRNA) recognizes a 20-nucleotide (nt) DNA sequence upstream of the 5′-NGG-3′ protospacer adjacent motif (PAM) and directs the DNA endonuclease Cas9 for site-specific cleavage (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013a). Based on this, we have developed an efficient genome-editing platform in hESCs, which we named iCRISPR (González et al., 2014). Through TALEN-mediated gene targeting in the AAVS1 locus, we have created hESC lines (referred to as iCas9 hESCs) that allow robust, doxycycline-inducible expression of Cas9. By transfecting iCas9 hESCs with gRNAs, the iCRISPR system enables efficient NHEJ-mediated gene disruption as well as HDR-mediated precise nucleotide modifications in the presence of short single-stranded DNA (ssDNA) donors (∼100 nt). We reasoned that the iCRISPR system would also facilitate the generation of knockin reporter alleles using longer double-stranded (dsDNA) donors and may further enable the identification of correctly targeted hESC lines without drug selection. Thus, this work explores the utility of iCRISPR for targeting fluorescent reporters into two endogenous loci, OCT4 (POU5F1) and PDX1, and demonstrates the generation of knockin hESC lines without drug selection for both expressed and silent genes. Further characterization confirmed the creation of multiple hESC reporter lines with no undesired mutations in the targeted loci or any potential off-target sites analyzed, supporting the broad application of this approach for efficient generation of knockin alleles in hESCs.