Name: genome editing and directed differentiation of hpscs for interrogating lineage determinants in human pancreatic development
Uploaded: 2017-03-05T15:00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development at JoVE.com

This work was funded in part by NIH/NIDDK (R01DK096239) and New York State Stem Cell Science (NYSTEM C029156). Z.Z. was supported by the NYSTEM postdoctoral fellowship from the Center for Stem Cell Biology of the Sloan Kettering Institute.

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The authors have nothing to disclose.

Time Considerations for Generating Mutant Lines
Although recent approaches based on CRISPR/Cas systems for genome editing have led to successful targeting, a more efficacious and universal platform would be preferable for larger-scale analyses of gene function. The iCRISPR platform offers a rapid and efficient method to introduce mutations to any gene of interest5,9. First, the PCR-based gRNA synthesis method allows the production of hundreds of gRNAs in arrayed format in one day without the time-consuming cloning steps. Second, with doxycycline-inducible Cas9 expression in iCas9 hPSCs, the step involving gRNA transfection requires only a minimal amount of work, and thus, multiple gRNA targeting experiments can be conducted concurrently. Third, due to the high targeting efficiencies attainable with our system, the analysis of ~ 24 - 48 colonies per gRNA transfected should be sufficient to establish multiple monoallelic and biallelic mutant lines for a single gene, although efficiencies vary depending on the target locus. Since it is feasible for a trained individual to mechanically pick 384 colonies (4 x 96-well plates) in one sitting, which should take ~ 4 h under a dissecting microscope, a trained individual can be expected to generate mutant lines affecting 12 genes within 1 - 2 months. The streamlined generation of hPSC mutants in a short time allows for the systematic analysis of an array of transcription factors and/or signaling pathway components that interact with each other, regulating the developmental process9. Additionally, efficient multiplexed gene targeting also opens the door to investigating genetic interactions underlying complex human traits.
Generation of Precise Genetic Modifications
The derivation of patient-specific iPSCs from easily accessible somatic cell types and the differentiation into disease-relevant cell types provide a great opportunity for the functional validation of disease-associated mutations. However, due to the considerable variability in genetic background between individuals, direct comparisons between iPSCs from patients and from healthy donors may not allow one to distinguish disease phenotypes from background effects. Therefore, it is necessary to generate isogenic control iPSCs by correcting the disease mutation back to the wild-type sequence or to introduce the patient-specific mutations into a wild-type hPSC background, as proposed by others25,26. In addition to loss-of-function (null) mutations, one may now more precisely dissect disease mechanisms through the introduction of a patient-specific sequence alterations into the endogenous locus in hPSCs, including hypermorphic, hypomorphic, neomorphic, or dominant-negative patient mutations.
Precise genetic modification employs HDR for DSB repair in the presence of a repair template. Since it is much less efficient than NHEJ-mediated DNA repair, a donor plasmid containing the patient-specific mutation, a drug selection cassette, and homology arms has been previously used as the repair template2. After drug selection and verification of the patient-specific mutation, a second step is generally needed to remove the drug selection cassette. While the most widely used Cre-loxP and FLP-FRT systems leave behind residual sequence in the endogenous locus, the use of piggyBac transposon has allowed for the seamless removal of the drug selection cassette27. More recently, short ssDNA templates have also been shown to support efficient HDR with engineered DNA endonucleases28. Compared to a donor plasmid, ssDNA can be directly synthesized and thus circumvents the time-consuming cloning step. Here, it has been shown that, by co-transfecting gRNA and an ssDNA template to induce homology-directed repair, iCRISPR can be used to introduce specific nucleotide modifications with high efficiency. This is critical for not only dissecting the role of essential nucleotides within protein functional domains, but also for modeling human disease mutations and potentially correcting these disease-associated mutations for therapeutic intervention. Due to the large number of susceptibility loci that are each associated with multiple sequence variants, modeling complex and multigenic diseases such as diabetes has been a challenge for geneticists. As the iCRISPR platform is permissive for the rapid generation of an allelic series or for multiplexable gene targeting, it can facilitate the investigation of multiple disease-associated loci, either individually or in combination with isogenic backgrounds.
Targeting Efficiencies and Off-target Effects
We have found a good correlation between the T7E1 and RFLP assay results and the number of mutant lines identified by sequencing. This emphasizes the importance of performing these assays in parallel with the establishment of clonal lines. While the targeting efficiencies attained may vary depending on the genomic loci, in most single-gene-targeting experiments, 20 - 60% of the clones were found with both alleles mutated (including in-frame and frameshift mutations)9. In cases where multiplexed gene targeting was performed, triple biallelic mutant clones with 5-10% efficiencies were obtained5. ssDNA-mediated HDR of several genes were also performed to obtain precise genetic alterations, with the efficiencies of obtaining homozygous knock-in clones ranging from 1 - 10%5. Working with CRISPR/Cas in hPSCs, any mutations in potential off-target sites that do not share the same gRNA target sequence are yet to be detected5,9,12. Whole-genome sequencing performed in a recent study has also failed to identify substantial off-target mutations in clonal hPSC lines generated using CRISPR/Cas29. Nevertheless, to minimize any potential effect of confounding phenotypes introduced by mutations at off-target effect sites, it is suggested to generate independent mutant lines using at least two independent gRNAs targeting different sequences within the same gene. Similar phenotypes observed in multiple lines generated using different gRNAs could principally rule out the possibility that the phenotype comes from an off-target effect.
Feeder-dependent versus Independent Culture and Targeting
Traditional methods for hPSC culture involve their maintenance and expansion on feeder cells in media containing serum or serum replacement that includes animal products, such as bovine serum albumin. Feeder cells, serum, serum replacement, and albumin all contain complex, undefined components and show considerable batch variability. Adapting to the feeder-free and chemically defined condition significantly reduces the efforts for hPSC maintenance and, more importantly, increases the consistency of differentiation experiments. At present, there are very few studies that describe genome editing procedures on hPSCs cultured in fully defined culture conditions30. We have found higher CRISPR targeting efficiencies when gRNA transfection and clonal deposition were performed in feeder-free conditions compared to feeder-dependent culture conditions. We believe that this is due to increased cell survival after transfection and single-cell seeding for colony formation. Moreover, feeder cells have previously been shown to sequester transfection reagents, thereby lowering the transfection efficiency.
Combining Genome Editing with Directed Differentiation
Previous differentiation protocols have yielded only a small fraction of insulin-positive cells, the majority of which were polyhormonal and resembled fetal endocrine cells23. Recent progress has permitted the differentiation of hPSCs into more mature glucose-responsive beta-like cells16,17,19,20. We can routinely obtain at least 75% definitive endoderm cells, 40% PDX1+NKX6.1+ pancreatic progenitors, and around 20% NKX6.1+CPEP+ glucose-responsive beta-like cells using HUES8 hPSCs9. When combined with the iCRISPR genome editing system, this more robust differentiation protocol has facilitated the analysis of transcription factors that are crucial to the pancreatic progenitor and endocrine stages of pancreatic differentiation. This will make it possible, in future studies, to examine a large number of candidate disease genes for the functional validation and investigation into the mechanisms that underlie diabetes9.
Future Applications or Directions
Our iCRISPR system may facilitate the generation of more complex genomic modifications, such as the creation of reporter alleles through HDR-mediated gene targeting using long donor DNA templates encoding protein tags or fluorescent reporters12. We have shown that, due to the high CRISPR targeting efficiencies attained in the system, this process can be performed in hPSCs, without the need for further drug selection12.&#160; Furthermore, multiplexed gene targeting can be used to investigate genetic interactions underlying complex human disease, as shown in our recent study, which we believe is the first example of such work31. iCRISPR can also be used to understand gene regulatory control by creating deletions either in noncoding RNAs or in gene regulatory regions, such as promoters and enhancers. To efficiently generate regulatory mutants using iCRISPR, gRNAs can be designed to disrupt the binding site of a DNA-binding protein, including but not restricted to the basal transcriptional machinery or a tissue-specific transcription factor. ssDNA template-mediated HDR can also be used to mutate specific protein-binding sites. Finally, we envisage that further optimization would enable the use of the iCRISPR platform in hPSCs for higher throughput genetic analyses of pluripotency phenotypes or disease phenotypes when combined with an in vitro differentiation protocol. These iCRISPR-mediated studies may allow for the more rapid identification of candidate disease-associated genes and studies of their functional relevance.

Human pluripotent stem cells (hPSCs) have the ability to both self-renew and give rise to all derivatives of the three embryonic germ lineages. They provide a valuable resource for cell replacement therapy and disease modeling by serving as a unique platform to recapitulate cellular processes in a human developmental context. They are also a source of experimental cells for scalable, high-throughput analyses. However, progress has been limited because of two main challenges: the lack of efficient genetic modification tools and the difficulty in recapitulating the complex embryonic developmental steps in a culture dish.
Genetic modification is an indispensable tool to study gene function in normal development and disease. However, while classical gene targeting approaches via homologous recombination have proven to be a powerful tool to dissect gene function in mouse embryonic stem cells (mESCs)1, this approach has been extremely inefficient when applied to hPSCs2,3. The recent brisk accession of programmable, site-specific nucleases from nature to laboratory use, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems4, means that genome engineering has become a much easier task in a wide range of organisms and cell lines, including in hPSCs. These gene editing tools take advantage of the fact that chimeric nucleases such as the Cas9 endonuclease can permit a whole range of genetic modifications by inducing double-stranded breaks (DSBs) at precise locations, triggering the endogenous DNA repair machinery to activate either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Both mechanisms may be exploited for genetic manipulation by inducing either random insertion and deletion mutations (indels; via NHEJ), to create frameshift mutations that nullify gene alleles, or precise nucleotide substitutions (via HDR), to recapitulate patient mutations for human disease modeling or to correct a disease-causing mutation for gene therapy.
CRISPR/Cas-mediated genome engineering requires two components: the constant RNA-guided Cas9 endonuclease necessary for DNA cleavage and a variable CRISPR RNA (crRNA) and trans-activating (tracrRNA) duplex that specifies DNA target recognition. The crRNA/tracrRNA duplex can be replaced with a single chimeric guide RNA (gRNA), which have been found to work more efficiently5,6,7. While the CRISPR/Cas9 system has been adapted to most experimental organisms and cell lines, the delivery and expression of Cas9 and gRNA varies significantly and needs to be further optimized to achieve efficient genome editing in many systems, including hPSCs8. An efficient genome-editing platform, iCRISPR, has been established in hPSCs5. In this system, a TALEN-mediated approach has been used to target both alleles of the &#34;transgene safe harbor locus&#34; AAVS1 in trans, one allele with a reverse tetracycline-controlled transactivator (M2rtTA) and the other with a tetracycline response element (TRE) driving the expression of Cas9 (iCas9) in the hPSCs. In established clonal lines (iCas9 hPSCs), Cas9 is highly expressed with doxycycline treatment. Meanwhile, due to its small size (100 nt), single or multiple gRNAs can be easily delivered into iCas9 hPSCs with high efficiency and can direct Cas9 for site-specific cleavage, enabling efficient NHEJ-mediated gene disruption, as well as HDR-mediated precise nucleotide modifications in the presence of short single-stranded DNA (ssDNA) donor templates. The iCRISPR system can be used to successfully generate a panel of disease-mimicking hPSC lines with biallelic (homozygous or compound heterozygous) or heterozygous loss-of-function mutations in important developmental genes5,9. While a number of groups have reported efficient gene editing using CRISPR/Cas in hPSCs, the success remains limited to a small number of technologically adept laboratories. The iCRISPR platform offers an efficient yet simple solution for routine gene editing by researchers of different skill levels, and it has already been used in a number of published studies by our group and others9,10,11,12. This approach has also been further extended to inducible silencing based on the expression of dCas9-KRAB13.
Along with the progress in genome-editing technology, significant improvements have also been achieved in hPSC maintenance and directed differentiation. Culture conditions for hPSCs have evolved from irradiated mouse embryonic fibroblast (iMEF) feeder-dependent to feeder-free conditions on defined extracellular matrix components, and from complex media formulations to chemically defined medium conditions14. Such improvements have reduced the variability in hPSCs due to batch-to-batch differences in iMEF preparation and knockout serum replacement components, and thus provide a more reproducible environment for hPSC differentiation. Meanwhile, improved knowledge of signaling pathways governing human embryonic development, as well as discoveries from high-throughput drug screenings, have led to improved differentiation protocols15,16,17,18. These protocols more closely mimic in vivo developmental steps and generate cell types that closely recapitulate their in vivo counterparts. For hPSC differentiation into the pancreatic lineage, initial protocols mimicked early pancreatic development relatively well but eventually generated polyhormonal &#946; cells that were of immature fetal phenotypes and responded poorly to glucose stimulation. Recent advancements16,17,19,20 have allowed for the generation of glucose-responsive pancreatic &#946;-like cells, which will enable us to investigate later events, such as the formation and the further maturation of monohormonal &#946; cells.
Here, we detail the application of genome-modified lines for the study of pancreatic development by combining the iCRISPR system with the hPSC-based in vitro differentiation platform towards glucose-responsive pancreatic &#946;-like cells. This coupling of powerful genome editing tools with an improved hPSC differentiation protocol not only offers the speed and scale necessary to meet the growing demand for validating disease causality, but also enables sophisticated genetic manipulations for further mechanistic investigations into transcriptional control underlying normal development and disease9.

<table border="0" cellpadding="0" cellspacing="0" width="847">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:368px;">Chemically defined medium (E8)</td>
			<td style="width:261px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">A1517001</td>
			<td style="width:87px;">Essential 8 basal medium and Essential 8 supplement included</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Truncated recombinant human form of vitronectin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK inhibitor Y-27632</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissociation reagent (TrypLE Select enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12563029</td>
			<td>1X, animal origin free, recombinant enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 Sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>10131035</td>
			<td>Geneticin Selective Antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puromycin dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>Puromycin Selective Antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Polymerase (Herculase II Fusion)</td>
			<td>Agilent Technologies</td>
			<td>600679</td>
			<td>PCR kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEGAshortscript T7 Transcription kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1354</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEGAclear Transcription Clean-Up Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1908</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Doxycycline hyclate</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985062</td>
			<td>Reduced Serum Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNeasy Blood &#38; Tissue Kit</td>
			<td>QIAGEN</td>
			<td>69504</td>
			<td>Genomic DNA extraction kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB 131 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BLAR medium</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Custom-made with a published formulation (Rezania et al., 2014)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine supplement (GlutaMAX)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaHCO3</td>
			<td>Thermo Fisher Scientific</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>LAMPIRE Biological Laboratories</td>
			<td>7500855</td>
			<td>Fatty acid free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GDF8</td>
			<td>PeproTech</td>
			<td>120-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR-99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>GSK-3 inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td>Vitamin C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FGF7</td>
			<td>R&#38;D Systems</td>
			<td>251-KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SANT1</td>
			<td>Tocris Bioscience</td>
			<td>1974</td>
			<td>Hedgehog inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Retinoic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDN</td>
			<td>Stemgent</td>
			<td>04-0019</td>
			<td>BMP inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IWP-2</td>
			<td>Tocris Bioscience</td>
			<td>3533</td>
			<td>Wnt antagonist&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ITS-X</td>
			<td>Thermo Fisher Scientific</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TPB</td>
			<td>EMD Millipore</td>
			<td>565740-1MG</td>
			<td>PKC activator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#8217;,5-Triiodo-L-thyronine (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ALK5i II</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-270-445</td>
			<td>ALK5 inhibitor II</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZnSO4</td>
			<td>Sigma-Aldrich</td>
			<td>Z0251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GSiXX</td>
			<td>EMD Millipore</td>
			<td>565789</td>
			<td>Gamma secretase inhibitor XX, NOTCH signaling inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Cys (N-acetyl cysteine)</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trolox</td>
			<td>EMD Millipore</td>
			<td>648471</td>
			<td>Vitamin E analogue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">R428</td>
			<td>Selleck Chemicals</td>
			<td>S2841</td>
			<td>AXL receptor tyrosine kinase inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24mm Transwell with insert</td>
			<td>Corning Life Sciences</td>
			<td>3414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Pulser Xcell Electroporation System</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4 cm Electroporation Cuvettes</td>
			<td>Bio-Rad</td>
			<td>1652081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AAVS1-TALEN-L</td>
			<td>Addgene</td>
			<td>59025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AAVS1-TALEN-R</td>
			<td>Addgene</td>
			<td>59026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AAVS1-Neo-M2rtTA</td>
			<td>Addgene</td>
			<td>60843</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AAVS1-Puro-iCas9</td>
			<td>Addgene</td>
			<td>58409</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 Endonuclease I</td>
			<td>NEB</td>
			<td>M0302L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer 2</td>
			<td>NEB</td>
			<td>B7002S</td>
			<td>NEBuffer 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Long oligonucleotide</td>
			<td>Eton Bioscience or IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SOX17 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1924</td>
			<td>1: 500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FOXA2 antibody</td>
			<td>Millipore</td>
			<td>07-633</td>
			<td>1: 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CXCR4-APC antibody</td>
			<td>R&#38;D Systems</td>
			<td>FAB170A</td>
			<td>1: 25</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF2419</td>
			<td>1: 500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NKX6.1 antibody</td>
			<td>DSHB</td>
			<td>F55A12</td>
			<td>1: 500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NKX2.2 antibody</td>
			<td>DSHB</td>
			<td>74.5A5</td>
			<td>1: 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NEUROD1 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1084</td>
			<td>1: 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>1: 2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C-peptide antibody</td>
			<td>DSHB</td>
			<td>GN-ID4-c</td>
			<td>1: 2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucagon antibody</td>
			<td>Sigma-Aldrich</td>
			<td>G2654</td>
			<td>1: 1000</td>
		</tr>
	</tbody>
</table>

genome editing and directed differentiation of hpscs for interrogating lineage determinants in human pancreatic development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Watch this Scientific Journal Video about Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development at JoVE.com

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Research

JoVE Journal

Genetics

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...