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>
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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>
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			<td height="20" style="height:20px;">pWS-TK6&#160;</td>
			<td>Addgene</td>
			<td>20350</td>
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			<td height="20" style="height:20px;">pKD3&#160;</td>
			<td>Addgene</td>
			<td>45604</td>
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			<td height="20" style="height:20px;">pKD46</td>
			<td>The Coli Genetic Stock Center, CGSC</td>
			<td>7634</td>
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			<td height="20" style="height:20px;">PGK-neo-bpA sequence&#160;</td>
			<td>Addgene</td>
			<td>13442</td>
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			<td height="20" style="height:20px;">Human BAC clones of target genes&#160;</td>
			<td>https://bacpac.chori.org</td>
			<td></td>
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			<td height="20" style="height:20px;">JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag</td>
			<td>Addgene</td>
			<td>43861</td>
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			<td height="20" style="height:20px;">MLM3636, Human-gRNA-ExpressionVector with U6 promoter&#160;</td>
			<td>Addgene</td>
			<td>43860</td>
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			<td height="20" style="height:20px;">pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)</td>
			<td>Addgene</td>
			<td>42335</td>
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		<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>
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			<td height="20" style="height:20px;">Perl</td>
			<td>http://www.perl.org/get.html</td>
			<td></td>
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			<td height="20" style="height:20px;">NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/</td>
			<td></td>
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			<td height="20" style="height:20px;">BLAT</td>
			<td colspan="2">https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</td>
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			<td height="20" style="height:20px;">sgRNA Primer synthesis</td>
			<td>Sigma</td>
			<td></td>
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			<td height="20" style="height:20px;">One shot Top10 Electrocomp E. Coli Competent cells</td>
			<td>Life Technologies</td>
			<td>C4040-50</td>
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			<td height="20" style="height:20px;">AccuPrime Pfx SupperMix</td>
			<td>Life Technologies</td>
			<td>12344-040</td>
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			<td height="20" style="height:20px;">Restriction enzymes</td>
			<td>NEB and Life Technologies</td>
			<td></td>
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			<td height="20" style="height:20px;">Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Researech</td>
			<td>D4007</td>
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			<td height="20" style="height:20px;">DNA quick extraction buffer&#160;</td>
			<td>Epicentre</td>
			<td>QE0905T</td>
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			<td height="20" style="height:20px;">Herculase II Fusion DNA Polymerases</td>
			<td>Agilent Technologies</td>
			<td>600675</td>
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			<td>Thermo Scientific</td>
			<td>R0611</td>
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			<td>Stem Cell Technologies</td>
			<td>05940</td>
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			<td height="20" style="height:20px;">UltraPure Agarose&#160;</td>
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			<td>16500-100</td>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">B49</td>
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			<td style="width:265px;">Life Technologies</td>
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			<td style="width:141px;">10566040</td>
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			<td style="width:141px;">10439</td>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">10828010</td>
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			<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>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">11140050</td>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">A1110501</td>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">17105-041</td>
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			<td style="width:265px;">Life Technologies</td>
			<td style="width:141px;">25200-056</td>
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			<td style="width:141px;">12760-013</td>
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			<td style="width:265px;">Millipore</td>
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			<td style="width:265px;">BD</td>
			<td style="width:141px;">354357</td>
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			<td style="width:265px;">Millipore</td>
			<td style="width:141px;">PMEF-HL</td>
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			<td style="width:265px;">LifeTechnologies</td>
			<td style="width:141px;">11811</td>
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			<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>
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			<td style="width:265px;">&#160;Moravek Biochemicals and Radiochemicals</td>
			<td style="width:141px;">&#160;&#160; M251</td>
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			<td height="20" style="height:20px;">Electroporator: Gene Pulser Xcell&#160;</td>
			<td style="width:265px;">Bio-Rad</td>
			<td style="width:141px;"></td>
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			<td style="width:265px;">Bio-Rad</td>
			<td style="width:141px;">165-2088</td>
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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 ...

efficient-generation-hipsc-neural-lineage-specific-knockin-reporters

Research

JoVE Journal

Developmental Biology

11.0K visualizações.  The University of Texas Health Science Center at Houston. 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.