Clustered Regularly Interspaced Short Palindromic Repeats Targeting Research Articles

Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.

CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/μl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.

CRISPR-based genome editing in primary human pancreatic islet cells

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

Detection of genome-edited cells by oligoribonucleotide interference-PCR.

Genome editing by engineered sequence-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for analysis of gene functions. Several techniques have been developed for detection of genome-edited cells, but simple, cost-effective, and positive detection methods remain limited. Recently, we developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which hybridization of an ORN with a complementary DNA sequence inhibits amplification across the sequence. Here, we investigated whether ORNi-PCR can be used to detect genome-edited cells. First, we showed that ORNs that hybridize to a CRISPR target site in the THYN1 locus inhibited amplification across the target site, but no longer inhibited amplification after the target site was edited, resulting in mismatches. Importantly, ORNi-PCR could distinguish even single-nucleotide differences. These features of ORNi-PCR enabled detection of genome-edited cells by positive PCR amplification. In addition, ORNi-PCR was successful in discriminating genome-edited cells from wild-type cells, and multiplex ORNi-PCR simultaneously detected indel mutations at multiple loci. However, endpoint ORNi-PCR may not be able to distinguish between mono- and bi-allelic mutations, which may limit its utility. Taken together, these results demonstrate the potential utility of ORNi-PCR for the screening of genome-edited cells.

CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants.

Chronic granulomatous disease (CGD) is characterized by defects in the production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for the treatment of CGD in human hematopoietic cells requires preclinical transplant models. The use of the lymphocyte-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized for targeted knockout of mouse Cybb on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting Cybb exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at least one Cybb allele based on sequence analysis of tail snip DNA. A female mouse heterozygous for a 235-bp deletion in Cybb exon 1 was bred to an NSG male to establish the X-CGD NSG mouse strain, NSG.Cybb[KO]. Resulting male offspring with the 235 bp deletion were found to be defective for production of ROS by neutrophils and other phagocytes, and demonstrated increased susceptibility to spontaneous bacterial and fungal infections with granulomatous inflammation. The establishment of the phagocyte-defective NSG.Cybb[KO] mouse model enables the in vivo assessment of gene and cell therapy strategies for treating CGD in human hematopoietic cell transplants without obfuscation by functional mouse phagocytes, and may also be useful for modeling other phagocyte disorders in humanized NSG mouse xenografts.

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called “CRISPRs.” Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

Phages Fight Back: Inactivation of the CRISPR-Cas Bacterial Immune System by Anti-CRISPR Proteins

Phage infection poses a major threat to bacterial survival, and bacteria have evolved many mechanisms to protect against it. One such system is the CRISPR-Cas system, which utilizes sequence memory to protect bacteria from phage infection. CRISPR-Cas is a highly specific adaptive defense mechanism that protects against invasion by all mobile genetic elements, including phages, plasmids, and conjugative elements [1,2]. CRISPRs were first recognized in Escherichia coli in 1987 [3] and have since been identified in the genomes of approximately 48% of eubacteria and 95% of archaea [4]. Their widespread occurrence highlights the important role they play in the evolution of microbial and phage genomes. CRISPR-Cas systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) separated by short “spacer” sequences, and CRISPR-associated (cas) genes. CRISPR systems are classified according to their gene complement and mechanism of action [2]. There are several different types (I, II, III) and subtypes (e.g., I-E, I-F) of CRISPR-Cas systems, most of which target DNA. The CRISPR system functions by incorporating one or more CRISPR spacers at the leader end of a CRISPR locus when a novel phage or other mobile genetic element infects the bacterial cell. These spacers are derived from the DNA of the invading phage, and their introduction into the CRISPR locus provides immunity to further infection by that phage. This adaptive immunity serves to protect the bacterial progeny; if the same phage is encountered in the future, the CRISPR-Cas system will be activated and the foreign genome destroyed. In turn, the phages mutate to evade CRISPR targeting, thereby creating a constant evolutionary battle between them and the bacteria they infect.

Construction of a gene knockout CHO cell line using a simple gene targeting method

Background Therapeutic antibodies have become an important focus of the biopharmaceutical industry. The Chinese hamster ovary (CHO) cell line is a major host for therapeutic antibody production. To construct productive CHO cell lines, two major transfection methods are commonly used, i.e., random integration and gene targeting. Random integration is a common method in which randomly integrated transgenes cause variation in antibody productivity because they are located in various chromosomal regions that affect transgene expression levels. Recently, gene-targeting methods, in which exogenous genes are inserted into a specific chromosomal region, have improved remarkably. Gene targeting is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Homologous sequences located on both sides of the exogenous gene are used. We used the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system as a gene-targeting method. The CRISPR-Cas9 system induces double-strand breaks (DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination [1]. Guide RNA hybridizes to a target integration site and induces Cas9 protein expression, leading to DSB. Finally, the Cas9 protein cuts genomic DNA. In this study, we constructed a simple genetargeting method in CHO cells using the CRISPR-Cas9 system in which CRISPR vectors induce DSBs and genetargeting vectors are inserted at the DSB site. In the conventional method, gene-targeting vectors should contain homology arms for effective recombination. In this study, we used the CRISPER system without homology arms for gene-targeted recombination. Materials and methods We constructed a CRISPR-Cas9 vector that expresses a guide RNA sequence targeting a region on chromosome O. Chromosome O was selected based on a previous classification of gene-amplified CHO cell chromosomes in order of decreasing size and assigned letters from A to T by fluorescence in situ hybridization (FISH) [2]. The CRISPR targeting sequence was determined from the BAC clone Cg0031N14, which contained the chromosome O sequence. Gene targeting vectors (pcDNA-GFP-DHFR) with or without target site homology arms were constructed from BAC clone Cg0031N14. The percentage of exogenous gene integration into chromosome O was determined by a FISH analysis. Total RNA extracted from E14Tg2a (mouse ES cells) was kindly provided by Dr. Tohru Kimura, Kitasato University, Kanagawa, Japan.

Abstract 1213: Endogenous p53 affinity tagging with CRISPR

Abstract Several antimetabolites used for cancer treatment, including hydroxyurea (HU) and pemetrexed (PTX), stabilize tumor suppressor protein p53, but fail to induce the transcription of p53 downstream target genes. To understand the underlying mechanism for the deficiency of stabilized p53, we are quantitatively characterizing p53 posttranslational modification and its interaction partners with mass spectrometry (MS). To facilitate the purification of endogenous p53, we established an HCT116 human colon carcinoma cell line, in which one allele of endogenous p53 was tagged with a classic Tandem Affinity Tag (TAP) at the C-terminus, using adenovirus associated virus (AAV) mediated homologous recombination (HR). The purification of p53 from this cell exhibited severe losses at each step, compromising analysis by MS. Because of this limited yield from TAP purification, we performed a systematic tagging of p53 with 3xFLAG, Strep-II, and HALO tags, and their combinations, at both N- and C-termini. The latest CRISPR (clustered regularly interspaced short palindromic repeats)/cas9 nuclease system provides major advantages for endogenous gene modification through HR. Efficient site-specific protein tagging (or mutation) requires a DNA break point in the immediate vicinity, limiting the choice of guide DNA (gDNA) positions possible. By extending the complementary sequence between CRISPR targeting RNA (crRNA) and trans-activating crRNA (tracrRNA) in the chimeric single guide RNA (sgRNA), we expanded the essential Protospacer Adjacent Motif (PAM) sequence of the S. pyogenes CRISPR II system from NGG to NAG with equal targeting efficiency. Cas9 nickase (both D10A, and H840A-N854A-N863A) generated more favorable HR rates compared to non-homologous end joining (NHEJ), while mitigating off-target effects, in comparison to wild type cas9. Utilizing our conditions, we achieved 2-8% (for different tags) endogenous p53 tagging in the background of 10-60% NHEJ from wild type cas9 and single sgRNA. With cas9 nickase and two sgRNA, we consistently achieved 0.2-2% specific p53 tagging in the background of 0.01-8% NHEJ, with higher efficiency for 5′ than 3′ single strand overhangs generated. We are currently generating single cell clones, selecting those tags most faithful to natural p53 expression levels, and testing p53 purification efficiencies with different tags. In conclusion, we optimized the CRISPR system, extensively characterized the individual steps in generating single cell clones for endogenous gene tagging, and will apply the optimal tag to purify p53 and its co-eluted binding partners for quantitative MS analysis. Citation Format: Chen Yang, Cortney L. Lawrence, Charles E. Lyons, Shirley M. Taylor, Richard G. Moran. Endogenous p53 affinity tagging with CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2015-1213

562. Modification of TCR Specificity by TALEN and CRISPR

Adoptive T-cell transfer of T-cells with transgenic high avidity T-cell receptors (TCR) holds a lot of promise as therapeutic approach, however comprising certain challenges. Endogenous and transferred TCR-chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. Designer nucleases like transcription activator-like effector nucleases (TALEN) and RNA-guided nucleases of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system introduce specific DNA double strand breaks (DSB) at their target sites. This ability can be exploited for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout. Alternatively, if a homology-containing donor template is provided, transgenes can be inserted into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled seven TALEN monomers and two guide RNAs targeting the constant regions of the TCR α-chain (TRAC) and the TCR β-chain (TRBC1 and TRBC2), respectively. Here we show the specific DSB induction by TALEN and CRISPR-Cas nucleases at their target sites, examined in K562 and T-cells using Cel1-assay and deep sequencing. Compared to nucleofection of TALEN-expressing plasmids, electroporation of mRNA considerably improved TCR knockout efficiency in primary T-cells. Using TRAC-TALEN and TRBC-TALEN mRNA, surface CD3 expression was successfully eliminated in about 75% and 60% of T-cells, respectively. The nuclease specificity was examined by identifying integrase-deficient lentiviral vector (IDLV)-marked DSB using LAM-PCR and deep sequencing. Although clustered IDLV integration sites were detected at the TALEN and CRISPR target sites, we did not detect any off-target activity, indicating high specificity. Additionally, K562 cells were nucleofected with TALEN-expressing plasmids and a donor template containing a GFP expression cassette flanked by 800bp homologous sequences to the TRAC locus. Thereby we were able to verify targeted gene addition through HDR of TALEN-mediated DSB in about 10% of treated cells. TALEN and CRISPR that show high efficiency and specificity to their target sequence will be used for generating T-cells with high avidity TCR.

Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene.

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

Abstract 3617: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CAS) system mediated endogenous CD19 gene knockout model in burkitt lymphoma

Abstract BACKGROUND: Pediatric Burkitt lymphoma (PBL) is an aggressive mature B-cell malignancy and is the most common non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH 2012). The survival of Pediatric Burkitt lymphoma (PBL) has significantly improved over the past 30 years through the introduction of short and intensive multi-agent chemotherapeutic regimens (Cairo et al., JCO, 2012). CD19 is an antigen expressed on the surface of cells of the B lineage, and is recognized as a potential target for immunotherapy. CD19 immunotherapy has been emerged as a promising approach for B cell malignancies (Porter/June et al., NEJM, 2011). However, the mechanism(s) and function of CD19 is still poorly understood in BL. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (CAS) genome engineering has been developed for precision targeted genome mutagenesis in vitro and in vivo for new experimental and therapeutic tools (Jao et al., PNAS, 2013; Morgan et al., Nat. Met., 2013; Fu et al., Nat. Biotech, 2013). OBJECTIVES: We hypothesize that 1) CRISPR/Cas system is suitable for silencing of CD19 expression in BL, and 2) CRISPR/Cas-mediated CD19 knockout model will be provide useful tool for understanding the mechanism and function or CD19 and the potential use of CD19 antibodies as therapeutic agents in BL. METHODS: Synthetic gRNAs targeting CD19 locus were constructed using modified CRISPR/Cas system from Hwang et al (Nat. Biotech, 2013) for CD19 knockout Raji cells. CRISPR-Cas/RNA-Guided Nuclease Plasmids were obtained from Addgene (ID: 43860 and 43861). Surveyor mutation detection assay was used for CD19 CRISPR validities and confirmed by sequencing analysis. Western blotting was performed for measurement of protein level and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional CD19 CRISPR (C1 and C2) targeting start (exon 1) and stop codon sequences (exon 14) of endogenous CD19 gene were generated. Mutated sequences by non-homologous end joining (NHEJ) were confirmed in CRISPR mediated CD19 knockout pool Raji cells. We successfully obtained stably CD19 knockout out single clone (hemizygote) which is entire CD19 gene (about 7.0kb, exon1 to exon 14) was deleted by CRISPR/Cas (Clone CD19-SC8). CD19-SC8 showed that a significant decrease of CD19 protein expression (92±0.2% reduction, p<0.0001) compared to empty vector-transfected Raji cells. CONCLUSIONS: This preliminary data suggest that CRISPR/Cas system is useful tool for the modification of endogenous CD19 gene expression and CRISPR/Cas mediated CD19 knockout model will be useful for studying CD19 function in BL and the efficacy of second generation CD19 antibodies and antibody conjugates against CD19 in BL. Citation Format: Sanghoon Lee, Changhong Yin, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CAS) system mediated endogenous CD19 gene knockout model in burkitt lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2014-3617

In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits.

The iCRISPR platform for rapid genome editing in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.