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 dual reporter cassette(s), with self-cleaving 2A peptide sequences (F2A, E2A or T2A) 20,21, or internal ribosome entry site (IRES). 
	Note: Reporters may include fluorescent proteins such as GFP, tdTomato, tagRFP, YFP, luciferase, or antibiotic resistance cassette, a combination of fluorescent protein and antibiotic resistance cassette. An example is shown in Figure 1 for constructing targeting vector for human ALDH1L1 gene, tagged by EGFP and neomycin resistance cassette.</li>
	<li>Add a positive selection cassette downstream of the reporter. The positive selection is a floxed antibiotic resistant cassette which will be excised from the targeted iPSCs after the positive clones are selected and isolated.</li>
	<li>Include a negative selection cassette outside of the 3&#8217; homology arm. Common negative selection cassettes are thymidine kinase, TK, and diphtheria toxin A (DTA) 22.</li>
	<li>Obtain a human BAC clone containing the sequence of the lineage marker and verify the clone by PCR amplification. Construct the targeting vector in pKD46 containing-DH5&#945; using recombineering to pull down the targeting site 23. Apply multisite gateway, Golden gate strategies23,24 when necessary.</li>
</ol>
2. Design and Vector Construction of sgRNAs for CRISPR/Cas9 System.
<ol>
	<li>Determine the lineage specific marker that will be targeted to make the reporter. Copy the cDNA sequence from NCBI database. Go to the human BLAT search to obtain genomic DNA alignment.</li>
	<li>Click the tab &#8220;browser&#8221; to view the assembly of the gene. Under &#8220;View&#8221; tab, click &#8220;DNA&#8221; to obtain the full genomic DNA sequence in FASTA format.</li>
	<li>Locate the stop codon within the DNA sequence, where the 2A sequence and the reporter cassette will be tagged.</li>
	<li>Search for the protospacer adjacent motif (PAM, i.e., NGG) within the vinicity (~100 bp flanking area) of the stop codon. Scan both strands to locate the appropriate NGG sequence. Consider the specificity of the upstream sequences to avoid potential off-target events. Use open source tools such as BLAST and CasOT (see Step 5 for details).</li>
	<li>Copy the sequence of 20 bases immediately upstream of NGG and paste it into the web designing tool ZiFit to design oligos with overhangs compatible with human-gRNA-expression vector (with U6 promoter) MLM3636.</li>
	<li>Synthesize both forward and reverse oligos 7.</li>
	<li>To make double-stranded sgRNA, denature both oligos at 70 &#176;C for 15 min, and gradually cool down to RT. For a 50 &#181;l reaction, add 22.5 &#181;l of forward and reverse oligos (stock concentration is 0.1 mM) respectively, and 5 &#181;l of annealing buffer (100 mM Tris-HCl, 10 mM EDTA, 1.5 M NaCl, pH 8.0).</li>
	<li>Mix and ligate the oligos obtained from step 2.7 with BsmBI-digested human-gRNA-expression vector (with U6 promoter) MLM3636. For a 20 &#181;l reaction, add 1 &#181;l of oligos from Step 2.7, 100 ng of MLM3636, and 1 &#181;l (400 U) of T4 ligase. Incubate at RT O/N.</li>
	<li>Transform DH5&#945; competent cells with the ligation product by heat shock at 42&#176;C for 30 sec. Grow the bacteria in LB agar Ampicillin (100 &#956;g/ml) plate at 37&#176;C O/N. Pick 3~5 colonies for plasmid miniprep according to manufacturer&#8217;s protocol. Sequence the plasmid DNA to confirm using commercial vendors. Maxiprep the correct plasmid according to manufacturer&#8217;s protocol.</li>
	<li>Evaluate sgRNA by T7 endonuclease 1 (T7E1) assay (see Step 4 for details). Transfect hiPSC with the plasmid and sgRNA will be expressed under the built-in U6 promoter in the MLM3636 backbone (See step 6 for details).</li>
</ol>
3. Design and Construction of Cas9n Double Nickase sgRNA
<ol>
	<li>Obtain double nickase Cas9n D10A vector pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A).</li>
	<li>Design a pair of forward and reverse oligos for each targeting locus. Copy the sequence of 20 bases immediately upstream of NGG as described in Step 2 and paste it into web designing tool ZiFit</li>
	<li>To obtain oligos with overhangs compatible with pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A), remove the first and last base of both forward and reverse oligos that are shown by the ZiFit designing tool.</li>
	<li>Synthesize forward and reverse oligos7. Name these oligos as f(n)-F, f(n)-R for PAMs on one side of the target site, r(n)-F, r(n)-R for PAMs on the other side of the target site. Of these, n is the number to designate different PAMs, lowercase f and r represent the location of the PAMs either at sense (f) or antisense (r) strand,, F and R represent forward and reverse oligos.
	<ol>
		<li>Test multiple pairs (e.g., r5 and f2) using the T7E1 assay (detailed in Step 4) to identify the optimized pair of sgRNAs to be used for Cas9n.</li>
	</ol>
	</li>
	<li>To make double-stranded sgRNA, denature both oligos at 70 &#176;C for 15 min, and gradually cool down to RT. For a 50 &#181;l reaction, add 22.5 &#181;l of forward and of reverse oligos (stock concentration is 0.1 mM) respectively, and 5 &#181;l of annealing buffer (100 mM Tris-HCl, 10 mM EDTA, 1.5 M NaCl, pH 8.0).</li>
	<li>Mix and ligate the oligos obtained from step 3.5 with BbsI-digested pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A). For a 20 &#181;l reaction, add 1 &#181;l of sgRNA from Step 3.5, 100 ng of Cas9n (D10A), 1 &#181;l (400 U) of T4 ligase. Incubate at RT O/N.</li>
	<li>Transform DH5&#945; competent cells with the ligation product by heat shock at 42 &#176;C for 30 sec. Grow the bacteria in LB agar Ampicillin (100 &#956;g/ml) plate at 37 &#176;C O/N. Pick 3~5 colonies for plasmid miniprep according to manufacturer&#8217;s protocol. Sequence the plasmid DNA to confirm using commercial vendors. Maxiprep the correct plasmid according to manufacturer&#8217;s protocol.</li>
	<li>Evaluate sgRNA by T7E1 assay (see Step 4 for details). Transfect hiPSC with the plasmid and sgRNA will be expressed under the built-in U6 promoter in the Cas9n (D10A) backbone (See step 6 for details).</li>
</ol>
4. Evaluation of sgRNAs by T7 Endonuclease1 (T7E1) Assay
<ol>
	<li>For one 24-well plate, plate 2 x 105 293FT cells per well one day prior (day -1).</li>
	<li>On the day of transfection (day 0), co-transfect 293FT cells with sgRNA plasmid and Cas9 expression vector. Add 0.5 &#181;g of sgRNA vector, 0.5 &#181;g of Cas9 expression vector JDS246 (Cas9-003), and 1.5 &#181;l of Lipofectamine 2000.
	<ol>
		<li>For negative control, add 0.5 &#181;g of GFP expression vector (e.g., CMV promoter driven GFP in a pcDNA3 backbone) instead of sgRNA vectors.</li>
	</ol>
	</li>
	<li>After 72 hr (day 3), harvest cells by adding 100 &#181;l of DNA quick extraction buffer and triturating vigorously.</li>
	<li>Incubate the mixture at 68 &#176;C for 15 min, then 95 &#176;C for 8 min. This is the template for the subsequent PCR experiments.</li>
	<li>Design both forward and reverse PCR primers to amplify the sequence flanking PAM. Usually the targeting PCR product is around 400-500 bp in length. Synthesize the oligos7.</li>
	<li>For a 50 &#181;l PCR reaction, add 1 &#181;l of template from Step 4.5, 0.5 &#181;l of Herculase II, 0.5 &#181;l of DMSO, 10 &#181;l of 5x buffer, 0.5 &#181;l of dNTP mix, and 1 &#181;l of forward and reverse primer mix.</li>
	<li>Use the following PCR parameters: 98 &#176;C for 2 min, 35 cycles of 98 &#176;C for 20 sec, 59 &#176;C for 20 sec, 72 &#176;C for 1 min, with a final extension at 72 &#176;C for 5 min. 
	Note: The PCR parameters described here yield specific products for several genes tested. However, these conditions are dependent upon user-chosen genes of interest. PCRs using genomic DNA as template often require optimization to ensure that no extra bands appear. These extra bands can be confused with cleavage products after the T7E1 assay.</li>
	<li>Measure DNA concentration of the PCR products using a spectrometer at A260.</li>
	<li>Add 400 ng of PCR products to 2 &#181;l of Digestion buffer 2. Add ddH2O to a final reaction volume of 19 &#181;l. Incubate in boiling water for 5 min. Let the reaction sit on the bench and naturally cool down to RT (45-60 min).</li>
	<li>Add 1 &#181;l (10 units/&#181;l) of T7E1, incubate at 37 &#176;C for 15 min. Stop the reaction by adding 2 &#181;l of 0.25 M EDTA mixed with 6x DNA Loading Dye.</li>
	<li>Dissolve 2.5 g of agarose powder to 100 ml of 1xTAE. Microwave the mixture and pour gel onto a gel box. When gel is formed, load the whole 20 &#181;l reaction to the 2.5% agarose gel. Run the gel at 100-150 V for 30 min.</li>
	<li>Inspect the gel under UV light (Figure 3). T7E1 cuts at the mismatched sites. For sgRNAs that have caused non-homologous end joining, the PCR products will show additional fast migrating bands (smaller in size) compared to negative control (described in step 4.3).</li>
	<li>Estimate and rank sgRNA cutting efficiency and insertion and deletion percentage (indel%) by comparing the band density using Image J software.
	<ol>
		<li>For each sample that run on the gel, designate the gel bands as uncut band, cut band 1 and cut band 2.</li>
		<li>Measure the density of each band using ImageJ according to the software handbook.</li>
		<li>Calculate fcut using the following formula: fcut = (cut band 1 + cut band 2) / (uncut band + cut band 1 + cut band 2)</li>
		<li>Calculate Indel% using the following formula: Indel=1- &#8730;(1-fcut).</li>
	</ol>
	</li>
</ol>
5. In Silico Prediction of Potential Off-target Sites
Note: Potential off target sites can be predicted using an online open source tool called CasOT17, which is a Perl based program.
<ol>
	<li>For a Windows system, download the CasOT program and the human genome database files to local hard drive.</li>
	<li>Prepare the target file of the designed sgRNA in the FASTA format.</li>
	<li>Open the file named &#8220;casot&#8221;. A command window appears. Leave it as is for now.</li>
	<li>Open the file named &#8220;opt-generator&#8221; to generate option string. Paste the path and name of the target file into the dialog of &#8220;-t&#8221; or &#8220;-target&#8221; option.</li>
	<li>Paste the path of the genome sequence file into the dialog of &#8220;-g&#8221; or &#8220;-genome&#8221; option.</li>
	<li>Input values of &#8211;s or &#8211;seed (default value is 2) and &#8211;n or -nonseed (default value is 255). The option string is generated and will appear in the box at the top of the page.</li>
	<li>Copy the option string and paste it into the CasOT command window described in Step 5.3. 
	Note: It might take 30-90 min to run the CasOT program depending on the options and the computer used. Once completed, a folder named &#8216;&#60;input_target_file&#62;-&#60;genome_file&#62;-&#60;off-target_related_options&#62;&#8217; will be generated. Two files will be included in the folder, a &#8220;.stat&#8221; file describing the statistics and a .csv (comma-separated values) file containing the detailed search results.&#60;/off-target_related_options&#62;&#60;/genome_file&#62;&#60;/input_target_file&#62;</li>
	<li>Open the .csv file with a spreadsheet program. The following information is provided in the file for every off target site: chromosomal and genomic location, sequence of the site, mismatch (Mm) type, total number of mismatched bases, and exon information relating to the off target site.</li>
	<li>Design PCR primers for the sites that have &#60;2 Mm in either the seed or the non-seed sequence.</li>
	<li>Perform PCR for selected positive hiPSC clone using the following conditions: 98 &#176;C for 2 min, 35 cycles of 98 &#176;C for 20 sec, 59 &#176;C for 20 sec, 72 &#176;C for 1 min, with a final extension at 72 &#176;C for 5 min. 
	Note: The PCR parameters described here yield specific products for several genes tested in our lab. However, these conditions are dependent upon user-chosen genes of interest. PCRs using genomic DNA as template often require optimization to ensure that no extra bands appear.</li>
	<li>Sequence the PCR products, align sequencing results with predicted sequences and search for mutations. If mutations exist, then off-target events are identified.</li>
</ol>
6. Electroporation of hiPSCs to Make Neural Lineage Reporters
<ol>
	<li>Culture hiPSCs in TeSR-E8 medium on Geltrex coated dishes. Change medium to MEF-CM (Mouse embryonic fibroblasts conditioned medium) medium two days before electroporation. Incubate in 2 ml of Accutase for 5 min and collect cells.</li>
	<li>Electroporate hiPSCs with linearized targeting vector, Cas9-003 expression vector, and sgRNA vector using the following conditions: 1 x 107 hiPSCs, co-transfect 2.5 &#181;g of sgRNA, 2.5 &#181;g of Cas9 and 50 &#181;g of targeting vector. For double nickase strategy, add 50 &#181;g of targeting vector, 2.5 &#181;g of each sgRNA (with Cas9n co-expressed by the vector).</li>
	<li>Select clones by positive and negative selections as described24,25.</li>
	<li>Identify positive clones by Southern blot analysis or long range genomic PCR24,25.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>pStart-K</td><td>Addgene</td><td>20346</td><td></td></tr><tr><td>pWS-TK6&amp;nbsp;</td><td>Addgene</td><td>20350</td><td></td></tr><tr><td>pKD3&amp;nbsp;</td><td>Addgene</td><td>45604</td><td></td></tr><tr><td>pKD46</td><td>The Coli Genetic Stock Center, CGSC</td><td>7634</td><td></td></tr><tr><td>PGK-neo-bpA sequence&amp;nbsp;</td><td>Addgene</td><td>13442</td><td></td></tr><tr><td>Human BAC clones of target genes&amp;nbsp;</td><td>https://bacpac.chori.org</td><td></td><td></td></tr><tr><td>JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag</td><td>Addgene</td><td>43861</td><td></td></tr><tr><td>MLM3636, Human-gRNA-ExpressionVector with U6 promoter&amp;nbsp;</td><td>Addgene</td><td>43860</td><td></td></tr><tr><td>pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)</td><td>Addgene</td><td>42335</td><td></td></tr><tr><td>sgRNA design, ZiFit</td><td>http://zifit.partners.org/ZiFiT/</td><td></td><td></td></tr><tr><td>Off target prediction tool CasOT</td><td>http://eendb.zfgenetics.org/casot/download.php</td><td></td><td></td></tr><tr><td>Perl</td><td>http://www.perl.org/get.html</td><td></td><td></td></tr><tr><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/</td><td></td><td></td></tr><tr><td>BLAT</td><td>https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</td><td></td><td></td></tr><tr><td>sgRNA Primer synthesis</td><td>Sigma</td><td></td><td></td></tr><tr><td>One shot Top10 Electrocomp E. Coli Competent cells</td><td>Life Technologies</td><td>C4040-50</td><td></td></tr><tr><td>AccuPrime Pfx SupperMix</td><td>Life Technologies</td><td>12344-040</td><td></td></tr><tr><td>Restriction enzymes</td><td>NEB and Life Technologies</td><td></td><td></td></tr><tr><td>Zymoclean Gel DNA Recovery Kit</td><td>Zymo Researech</td><td>D4007</td><td></td></tr><tr><td>DNA quick extraction buffer&amp;nbsp;</td><td>Epicentre</td><td>QE0905T</td><td></td></tr><tr><td>Herculase II Fusion DNA Polymerases</td><td>Agilent Technologies</td><td>600675</td><td></td></tr><tr><td>Digestion buffer 2</td><td>New England Biolab</td><td></td><td></td></tr><tr><td>6X DNA Loading Dye</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>TeSR-E8 Kit for hESC/hiPSC Maintenance</td><td>Stem Cell Technologies</td><td>05940</td><td></td></tr><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Life Technologies</td><td>16500-100</td><td></td></tr><tr><td>50xTAE</td><td>Life Technologies</td><td>B49</td><td></td></tr><tr><td>Essential 8 medium&amp;nbsp;</td><td>Life Technologies</td><td>A1517001</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline without Calcium and Magnesium&amp;nbsp;</td><td>Life Technologies</td><td>A12856-01</td><td></td></tr><tr><td>D-MEM/F12 with Glutamax&amp;nbsp;</td><td>Life Technologies</td><td>10565018</td><td></td></tr><tr><td>D-MEM with Glutamax&amp;nbsp;</td><td>Life Technologies</td><td>10566040</td><td></td></tr><tr><td>Fetal Bovine Serum-ES cell qualified&amp;nbsp;</td><td>Life Technologies</td><td>10439</td><td></td></tr><tr><td>Knockout serum replacement&amp;nbsp;</td><td>Life Technologies</td><td>10828010</td><td></td></tr><tr><td>2-mercaptoethanol 1000X&amp;nbsp;</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Non Essential Amino Acid&amp;nbsp;</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>Stempro Accutase&amp;nbsp;</td><td>Life Technologies</td><td>A1110501</td><td></td></tr><tr><td>Dispase&amp;nbsp;</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>0.25% Trypsin- EDTA solution&amp;nbsp;</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>12760-013</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Millipore</td><td>&amp;nbsp;&amp;nbsp; SCM075</td><td></td></tr><tr><td>SMC4 reagent&amp;nbsp;</td><td>BD</td><td>354357</td><td></td></tr><tr><td>Neomycin resistant MEF&amp;nbsp;</td><td>Millipore</td><td>PMEF-NL</td><td></td></tr><tr><td>Hygromycin resistant MEF&amp;nbsp;</td><td>Millipore</td><td>PMEF-HL</td><td></td></tr><tr><td>G418 (Geneticin)</td><td>LifeTechnologies</td><td>11811</td><td></td></tr><tr><td>Hygromycin B&amp;nbsp;</td><td>Life Technologies</td><td>10687010</td><td></td></tr><tr><td>FIAU (Fialuridine, 1-2-Deoxy-2-fluoro-&amp;szlig;-D-arabinofuranosyl-5-iodouracil</td><td>&amp;nbsp;Moravek Biochemicals and Radiochemicals</td><td>&amp;nbsp;&amp;nbsp; M251</td><td></td></tr><tr><td>Electroporator: Gene Pulser Xcell&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>0.4 cm electroporation cuvette&amp;nbsp;</td><td>Bio-Rad</td><td>165-2088</td><td></td></tr><tr><td>DIG-High prime DNA labeling and detection starter kit II&amp;nbsp;</td><td>Roche</td><td>11585614910</td><td></td></tr><tr><td>Hybridization denature solution&amp;nbsp;</td><td>VWR</td><td>82021-478</td><td></td></tr><tr><td>PCR DIG probe synthesis kit&amp;nbsp;</td><td>Roche</td><td>11636090910</td><td></td></tr><tr><td>DIG wash set&amp;nbsp;</td><td>Roche</td><td>11585762001</td><td></td></tr><tr><td>Anti-Digoxigenin (DIG-AP)</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>CSPD chemiluminescence system&amp;nbsp;</td><td>Roche</td><td>11755633001</td><td></td></tr><tr><td>DIG wash and block buffer set&amp;nbsp;</td><td>Roche</td><td>11585762001</td><td></td></tr><tr><td>50X TAE buffer&amp;nbsp;</td><td>Life Technologies</td><td>24710030</td><td></td></tr><tr><td>Blotting buffer (25 mM Tris pH 7.4,&amp;nbsp; 0.15 M NaCl,&amp;nbsp; 0.1% Tween20)</td><td></td><td></td><td></td></tr><tr><td>Hoefer Ultraviolet Crosslinker&amp;nbsp;</td><td>Fisher Scientific</td><td>03-500-308</td><td></td></tr><tr><td>Spermidine&amp;nbsp;</td><td>Fisher</td><td>AC13274-0010</td><td></td></tr><tr><td>Tris HCl 2M (pH 7.5)</td><td>VWR</td><td>&amp;nbsp;&amp;nbsp; 200064-506</td><td></td></tr><tr><td>Denville Scientific blue bio film 8x10&amp;nbsp;</td><td>Fisher</td><td>nc9550782</td><td></td></tr><tr><td>DNA molecular weight marker II ( DIG-labeled )</td><td>Roche</td><td>11218590910</td><td></td></tr><tr><td>Amersham Blotting membrane Hybond-N+&amp;nbsp;</td><td>Roche</td><td>95038-400</td><td></td></tr><tr><td>Pyrex glass drying tray&amp;nbsp;</td><td>Fisher</td><td>15-242A</td><td></td></tr><tr><td>Kimberly-Clark C-fold paper towels&amp;nbsp;</td><td>Fisher</td><td>06-666-32B</td><td></td></tr><tr><td>Whatman 3MM paper (26X41)</td><td>Fisher</td><td>&amp;nbsp;&amp;nbsp; 05-713-336</td><td></td></tr><tr><td>Hybridization bag&amp;nbsp;</td><td>Roche</td><td>11666649001</td><td></td></tr><tr><td>Hybridization tubes&amp;nbsp;</td><td>Fisher</td><td>13-247-300</td><td></td></tr><tr><td>Hybridization oven rotisserie Shake &#039;n&#039; Stack&amp;nbsp;</td><td>Fisher</td><td>HBMSOV14110</td><td></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...

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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 Views.  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.

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

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.

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 ...

Genetics

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Bioengineering

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Immunology and Infection

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

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

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