Name: efficient generation of hipsc neural lineage specific knockin reporters using the crispr/cas9 and cas9 double nickase system
Uploaded: 2015-05-28T15:00:00
Description: Watch this Scientific Journal Video about Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System at JoVE.com

This work was supported by the Department of Neurosurgery, Memorial Hermann Foundation Staman Ogilvie Fund, the Bentsen Stroke Center at the University of Texas Health Science Center at Houston, and Mission Connect TIRR Foundation.

1. Design and Vector Construction for Targeting Vectors
<ol>
	<li>Once the lineage specific marker is determined, locate the genomic sequence of the gene and design the homology arms accordingly. The length of 5&#8217; homology arm is ~1 kb and of 3&#8217; homology arm is ~1.5 kb (Figure 1).</li>
	<li>Tag the reporter cassette sequentially downstream of the genomic sequence right before the stop codon, by subcloning, so that the endogenous expression is not altered or disrupted. Link the reporter, or d...

<ol>
	<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+generation+of+mice+carrying+mutations+in+multiple+genes+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.</a> Cell. 153, 910-918 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Hwang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+editing+in+zebrafish+using+a+CRISPR-Cas+system.">Efficient genome editing in zebrafish using a CRISPR-Cas system.</a> Nature. 31, 227-229 (2013).</li><li>Friedland, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+genome+editing+in+C.+elegans+via+a+CRISPR-Cas9+system.">Heritable genome editing in C. elegans via a CRISPR-Cas9 system.</a> Nat Methods. , (2013).</li><li>DiCarlo, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+in+Saccharomyces+cerevisiae+using+CRISPR-Cas+systems.">Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.</a> Nucleic Acids Res. 41, 4336-4343 (2013).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339, 819-823 (2013).</li><li>Anders, C., Niewoehner, O., Duerst, A., Jinek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+PAM-dependent+target+DNA+recognition+by+the+Cas9+endonuclease.">Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.</a> Nature. 513, 569-573 (2014).</li><li>Deltcheva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+RNA+maturation+by+trans-encoded+small+RNA+and+host+factor+RNase+III.">CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.</a> Nature. 471, 602-607 (2011).</li><li>Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9-crRNA+ribonucleoprotein+complex+mediates+specific+DNA+cleavage+for+adaptive+immunity+in+bacteria.">Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, E2579-E2586 (2012).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337, 816-821 (2012).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-programmed+genome+editing+in+human+cells.">RNA-programmed genome editing in human cells.</a> eLife. 2, e00471 (2013).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+off-target+mutagenesis+induced+by+CRISPR-Cas+nucleases+in+human+cells.">High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.</a> Nature Biotechnology. 31, 822-826 (2013).</li><li>Hsu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31, 827-832 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAS9+transcriptional+activators+for+target+specificity+screening+and+paired+nickases+for+cooperative+genome+engineering.">CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.</a> Nature Biotechnology. 31, 833-838 (2013).</li><li>Pattanayak, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+profiling+of+off-target+DNA+cleavage+reveals+RNA-programmed+Cas9+nuclease+specificity.">High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.</a> Nature Biotechnology. 31, 839-843 (2013).</li><li>Shen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+modification+by+CRISPR-Cas9+nickase+with+minimal+off-target+effects.">Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.</a> Nature Methods. 11, 399-402 (2014).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154, 1380-1389 (2013).</li><li>Yang, H., Wang, H., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+genetically+modified+mice+using+CRISPR/Cas-mediated+genome+engineering.">Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.</a> Nature Protocols. 9, 1956-1968 (2014).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154, 1370-1379 (2013).</li><li>Szymczak, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nature Biotechnology. 22, 589-594 (2004).</li><li>Carey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+murine+and+human+somatic+cells+using+a+single+polycistronic+vector.">Reprogramming of murine and human somatic cells using a single polycistronic vector.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 157-162 (2009).</li><li>Yagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+negative+selection+for+homologous+recombinants+using+diphtheria+toxin+A+fragment+gene.">A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene.</a> Analytical Biochemistry. 214, 77-86 (1993).</li><li>Wu, S., Ying, G., Wu, Q., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+constructing+gene+targeting+vectors:+generating+knockout+mice+for+the+cadherin+family+and+beyond.">A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond.</a> Nature Protocols. 3, 1056-1076 (2008).</li><li>Xue, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+targeted+neuroglial+reporter+line+generated+by+homologous+recombination+in+human+embryonic+stem+cells.">A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.</a> Stem Cells. 27, 1836-1846 (2009).</li><li>Liu, Y., Jiang, P., Deng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OLIG+gene+targeting+in+human+pluripotent+stem+cells+for+motor+neuron+and+oligodendrocyte+differentiation.">OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation.</a> Nature Protocols. 6, 640-655 (2011).</li></ol>

Gene targeting is an essential tool in characterizing gene functions. However, the relatively low efficiency demands labor-intensive and time-consuming work. Recently development on genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has greatly improved the targeting efficiency. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps ...

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, together with other genome editing tools, has revolutionized the human genome manipulation in recent years1-7. Major components in the CRISPR/Cas9 system are single guide RNA (sgRNA) and Cas9. Cas9 is an RNA-guided type II DNA endonuclease. It has two nuclease domains, HNH and RuvC, which are responsible for making DNA double-stranded breaks (DSBs) at a specific genomic region next to a protospacer adjacent motif (PAM)7-10.An sgRNA has a combined function of that of crRNA and tracrRNA, two adaptive immunity RNA molecules identified in bacteria (such as Streptococcus pyogenes) and archaea to fight against DNA invasion of exogenous sources9-11. When appropriately designed and co-introduced to mammalian cells, the sgRNA recognizes the PAM sequence, base-pairs with the complementary strand of target DNA, and guides and transactivates Cas9 to cleave at both DNA strands immediately upstream of PAM7. Subsequently, homologous directed recombination (HDR, in the presence of a targeting vector) or non-homologous end joining (NHEJ) will occur to repair the DSBs. Transgenes such as cDNAs, reporter cassettes, or antibiotic resistant fragments can be integrated, and transgenic or knockin lines made.
Although the CRISPR/Cas9 system is highly efficient and relatively specific, undesired off-target activities have been reported12-15. To minimize off-target events, Cas9 nickases (Cas9n) have also been engineered9,10,14,16,17. Cas9n (Cas9 D10A or Cas9 H840A) is Cas9 enzyme with mutations at one of the two nuclease domains, which only elicits a nick at one strand of DNA. To achieve cleavage at both strands, two sgRNAs are designed to guide a pair of nickases, which cuts separately at nearby loci of the two different DNA strands. The &#8220;double nicking&#8221; strategy requires a more stringent design than the conventional Cas9 platform, and offers both high efficiency and high specificity for gene editing experiments.
This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporter using CRISPR/Cas system assisted homologous recombination. Steps for obtaining the necessary components for making neural lineage reporter lines using the CRISPR/Cas system mediated genome editing are described, with a focus on construction of targeting vectors and sgRNAs. This protocol has been successfully repeated in multiple hiPSC lines including those derived from healthy individuals and from patients with CNS diseases. Genes targeted in our laboratory include OLIG2, HB9 (MNX1), NEUROG2, SOX1, ALDH1L1 and SOD1. Targeting efficiency from CRISPR/Cas system mediated homologous recombination in hiPSCs ranges from 20-40%, which is consistent with previous reports1,18,19. Compared to a typical efficiency of 1-2% in conventional gene targeting experiments, the targeting efficiency is significantly improved.

<table border="0" cellpadding="0" cellspacing="0" width="664">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Name of Material/Equipment</td>
			<td style="width:265px;">Vendor</td>
			<td style="width:141px;">Catalog no.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pStart-K</td>
			<td>Addgene</td>
			<td>20346</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pWS-TK6&#160;</td>
			<td>Addgene</td>
			<td>20350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pKD3&#160;</td>
			<td>Addgene</td>
			<td>45604</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pKD46</td>
			<td>The Coli Genetic Stock Center, CGSC</td>
			<td>7634</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PGK-neo-bpA sequence&#160;</td>
			<td>Addgene</td>
			<td>13442</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human BAC clones of target genes&#160;</td>
			<td>https://bacpac.chori.org</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag</td>
			<td>Addgene</td>
			<td>43861</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MLM3636, Human-gRNA-ExpressionVector with U6 promoter&#160;</td>
			<td>Addgene</td>
			<td>43860</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)</td>
			<td>Addgene</td>
			<td>42335</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sgRNA design, ZiFit</td>
			<td>http://zifit.partners.org/ZiFiT/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Off target prediction tool CasOT</td>
			<td colspan="2">http://eendb.zfgenetics.org/casot/download.php</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perl</td>
			<td>http://www.perl.org/get.html</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BLAT</td>
			<td colspan="2">https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sgRNA Primer synthesis</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">One shot Top10 Electrocomp E. Coli Competent cells</td>
			<td>Life Technologies</td>
			<td>C4040-50</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AccuPrime Pfx SupperMix</td>
			<td>Life Technologies</td>
			<td>12344-040</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Restriction enzymes</td>
			<td>NEB and Life Technologies</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Researech</td>
			<td>D4007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA quick extraction buffer&#160;</td>
			<td>Epicentre</td>
			<td>QE0905T</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Herculase II Fusion DNA Polymerases</td>
			<td>Agilent Technologies</td>
			<td>600675</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digestion buffer 2</td>
			<td>New England Biolab</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6X DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8 Kit for hESC/hiPSC Maintenance</td>
			<td>Stem Cell Technologies</td>
			<td>05940</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure Agarose&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td>16500-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50xTAE</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">B49</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Essential 8 medium&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">A1517001</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:259px;">Dulbecco&#8217;s Phosphate Buffered Saline without Calcium and Magnesium&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">A12856-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">D-MEM/F12 with Glutamax&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10565018</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">D-MEM with Glutamax&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10566040</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Fetal Bovine Serum-ES cell qualified&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10439</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Knockout serum replacement&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10828010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">2-mercaptoethanol 1000X&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">21985023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Non Essential Amino Acid&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">11140050</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Stempro Accutase&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">A1110501</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Dispase&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">17105-041</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">0.25% Trypsin- EDTA solution&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">25200-056</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Geltrex&#160;</td>
			<td style="width:265px;">Life Technologies&#160;&#160;&#160;</td>
			<td style="width:141px;">12760-013</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">ROCK inhibitor Y-27632</td>
			<td style="width:265px;">Millipore</td>
			<td style="width:141px;">&#160;&#160; SCM075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">SMC4 reagent&#160;</td>
			<td style="width:265px;">BD</td>
			<td style="width:141px;">354357</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Neomycin resistant MEF&#160;</td>
			<td style="width:265px;">Millipore</td>
			<td style="width:141px;">PMEF-NL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hygromycin resistant MEF&#160;</td>
			<td style="width:265px;">Millipore</td>
			<td style="width:141px;">PMEF-HL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">G418 (Geneticin)</td>
			<td style="width:265px;">LifeTechnologies</td>
			<td style="width:141px;">11811</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hygromycin B&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10687010</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:259px;">FIAU (Fialuridine, 1-2-Deoxy-2-fluoro-&#223;-D-arabinofuranosyl-5-iodouracil</td>
			<td style="width:265px;">&#160;Moravek Biochemicals and Radiochemicals</td>
			<td style="width:141px;">&#160;&#160; M251</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator: Gene Pulser Xcell&#160;</td>
			<td style="width:265px;">Bio-Rad</td>
			<td style="width:141px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">0.4 cm electroporation cuvette&#160;</td>
			<td style="width:265px;">Bio-Rad</td>
			<td style="width:141px;">165-2088</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">DIG-High prime DNA labeling and detection starter kit II&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11585614910</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hybridization denature solution&#160;</td>
			<td style="width:265px;">VWR</td>
			<td style="width:141px;">82021-478</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">PCR DIG probe synthesis kit&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11636090910</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">DIG wash set&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11585762001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Anti-Digoxigenin (DIG-AP)</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11093274910</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">CSPD chemiluminescence system&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11755633001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">DIG wash and block buffer set&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11585762001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">50X TAE buffer&#160;</td>
			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">24710030</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Blotting buffer (25 mM Tris pH 7.4,&#160; 0.15 M NaCl,&#160; 0.1% Tween20)</td>
			<td style="width:265px;"></td>
			<td style="width:141px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hoefer Ultraviolet Crosslinker&#160;</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:141px;">03-500-308</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Spermidine&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">AC13274-0010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Tris HCl 2M ( pH 7.5 )</td>
			<td style="width:265px;">VWR</td>
			<td style="width:141px;">&#160;&#160; 200064-506</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Denville Scientific blue bio film 8x10&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">nc9550782</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">DNA molecular weight marker II ( DIG-labeled )</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11218590910</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Amersham Blotting membrane Hybond-N+&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">95038-400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Pyrex glass drying tray&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">15-242A</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Kimberly-Clark C-fold paper towels&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">06-666-32B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Whatman 3MM paper ( 26X41)</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">&#160;&#160; 05-713-336</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hybridization bag&#160;</td>
			<td style="width:265px;">Roche</td>
			<td style="width:141px;">11666649001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Hybridization tubes&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">13-247-300</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Hybridization oven rotisserie Shake &#39;n&#39; Stack&#160;</td>
			<td style="width:265px;">Fisher</td>
			<td style="width:141px;">HBMSOV14110</td>
		</tr>
	</tbody>
</table>

efficient generation of hipsc neural lineage specific knockin reporters using the crispr/cas9 and cas9 double nickase system

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Targeting vectors with all necessary components including homology arms, reporter cassette, positive and negative selection cassettes are constructed (Figure 1). Based on the endogenous genomic locus, multiple (2 to 3) sgRNAs (if using the Cas9 system) or sgRNA pairs (if using the Cas9n double nickase strategy) are designed and constructed into human-gRNA-expression vector MLM3636 or pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A) backbones (Figure 2). Before transfecting hiPSCs, the sgRNAs are...

Watch this Scientific Journal Video about Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System at JoVE.com

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...