We thank Kosuke Yusa for sharing the pMCS-AAT_PB-PGKpuroTK and pCMV-hyPBase plasmids. We thank Jia-Yin Yang and Karamjit Singh-Dolt for helpful discussions on genome editing. D.C.H. lab is supported by an award from the Chief Scientist Office (TC/16/37) and the UK Regenerative Medicine Platform (MR/L022974/1). Y.W. was supported by a PhD scholarship funded by the Chinese Scholarship Council and the University of Edinburgh.

1. Designing and constructing CRISPR/Cas9n-sgRNA expression plasmids
<ol>
	<li>Search for 20-bp guide sequences directly upstream of any 5&#39;-NGG near the site to be modified. A pair of single-guide RNAs (sgRNAs) is needed when the Cas9 nickase (Cas9n) from Streptococcus pyogenes is used. The pair of sgRNAs must be able to generate 5&#39; overhangs upon nicking with a -4 to 20 bp offset6.</li>
	<li>Order necessary oligos and pSpCas9n-2A-puro vector.</li>
	<li>Clone sgRNA into the pSpCas9n vector for co-expression with Cas9n following the published protocol12.</li>
</ol>
2. Designing and constructing a piggyBac-based targeting vector
<ol>
	<li>Download the target gene sequence and search for a TTAA site near the site to be modified. 
	NOTE: The distance between the TTAA site and the to-be-modified site should be as small as possible. If within coding sequence and no TTAA site is present within 100-bp distance, it is necessary to introduce one by making synonymous nucleotide substitutions.</li>
	<li>Design homology arms (HAs) and incorporate desired point mutations in the HAs. The optimal length of HAs should be around 500 - 1200 bp. 
	NOTE: It is recommended to introduce synonymous nucleotide substitutions to mutate the PAM sequence in order to avoid potential Cas9n re-cleavage.</li>
	<li>Construct the final targeting vector following an established protocol10. 
	NOTE: In the targeting vector used here, a PGK promoter driven puro-deltaTK selection cassette is flanked by transposon inverted repeats.</li>
</ol>
3. Genetic editing of human pluripotent stem cells
<ol>
	<li>Maintain human pluripotent stem cells (hPSCs) in a humidified incubator at 37 &#176;C and 5% CO2 on recombinant laminin 521-coated surfaces (5 &#181;g/mL) in mTeSR1 medium13. The cells should reach 70-85% confluency after 72 hours following a 1:3 split ratio. 
	NOTE: If present, remove spontaneously differentiated cells before daily medium change by gently aspirating them away.</li>
	<li>On the day of transfection, coat enough wells of a 24-well plate with 300 &#181;L of the laminin 521 solution (5 &#181;g/mL) at 37 &#176;C for at least 2 h.</li>
	<li>Remove the coating solution gently and add 300 &#181;L of fresh mTeSR1 medium supplemented with 10 &#181;M ROCK inhibitor to each well, and then put the plate back to the incubator to receive cells. 
	NOTE: Do not allow the plates to dry at any point when the laminin coating solution is removed.</li>
	<li>Move the stock hPSCs from the incubator, remove spent medium, and then wash the cells once using 1 mL of sterile 1x DPBS.</li>
	<li>Add 1 mL of gentle cell dissociation reagent to each well of a 6-well plate and incubate at 37 &#176;C for 6-8 min to dissociate the cells. 
	NOTE: Gently tap the plate and check whether the cells can detach easily to decide whether the digestion is long enough.</li>
	<li>Gently pipette up and down using a P1000 tip to lift off all the hPSCs.</li>
	<li>Terminate the dissociation by adding 2 mL of fresh mTeSR1 medium supplemented with 10 &#181;M ROCK inhibitor (Y-27632) to the cell suspension.</li>
	<li>Mix well and transfer the single-cell suspension to a 50 mL tube, and centrifuge at 200 x g for 3 min at room temperature.</li>
	<li>Remove the supernatant, and resuspend the cells well in 2 mL fresh mTeSR1 medium supplemented with 10 &#181;M ROCK inhibitor.</li>
	<li>Count the viable cells using a hemocytometer. Use Trypan Blue to stain and exclude dead cells.</li>
	<li>Transfer 8 x 105 to 1 x 106 live cells to a 1.5 mL tube for each nucleofection reaction and centrifuge at 200 x g for 3 min. Then carefully aspirate the supernatant.</li>
	<li>Mix 3 &#181;g of the paired Cas9n-sgRNA expression plasmids and 5 &#181;g of targeting vector plasmid in 100 &#181;L of mixed nucleofection solution/supplement from the human stem cell nucleofection kit. Use the GFP control plasmid from the kit and prepare a GFP control plasmid mix as well. 
	NOTE: It is important to make a maxi-prep of all plasmids to reduce endotoxin contamination before nucleofection. The concentration of the plasmids should be approximately 1 &#181;g/&#181;L.</li>
	<li>Use the DNA mix (volume ~111 &#181;L) to resuspend the prepared cells and transfer it to an electroporation cuvette (provided with the nucleofection kit), avoiding any bubbles.</li>
	<li>Electroporate the cells using the nucleofection device by selecting the optimized conditions for human pluripotent stem cells. 
	NOTE: If using the nucleofection kit for human pluripotent stem cells for the first time, it is necessary to test the electroporation program and choose the most efficient one.</li>
	<li>Immediately add 500 &#181;L of fresh and warm to 37 &#176;C mTeSR1 medium supplemented with 10 &#181;M ROCK inhibitor to the electroporated cells. Transfer the mix to 2 wells of a 24-well plate prepared from steps 3.2 and 3.3.</li>
	<li>Quickly put the plate back to the 37 &#176;C/5% CO2 incubator and allow the cells to recover.</li>
	<li>12-16 h later, change cell maintenance medium. If the cells established cell-cell contact, withdraw the ROCK inhibitor, if not, continue supplementing the medium with the inhibitor.</li>
	<li>Between 24-48 h, check the nucleofection efficiency by examining GFP expression in the control cells. GFP positive cells should be at least 30 percent.</li>
	<li>48 h after nucleofection, start selecting cells by supplementing the mTeSR1 medium with 1 &#181;g/mL puromycin.</li>
	<li>72 h after nucleofection, supplement the mTeSR1 medium with 0.5 &#181;g/mL puromycin. If the cell confluency is lower than 30%, also supplement the medium with 10 &#181;M ROCK inhibitor.</li>
	<li>4-6 days post-nucleofection, passage the puromycin-resistant cells to 10-15 x 96-well plates at the concentration of 0.8 cells/well. 
	NOTE: It is essential to supplement the medium with 10 &#181;M ROCK inhibitor and 0.5 &#181;g/mL puromycin.</li>
	<li>Maintain those cells at 37 &#176;C/10% CO2 for 10-12 days to form single cell-derived colonies. Top up medium once 7 days after seeding. 
	NOTE: Increased CO2 level helps single hPSCs to form colonies from our experience.</li>
	<li>Mark wells containing a single colony and replace the medium with fresh mTeSR1 medium containing 0.5 &#181;g/mL puromycin but no ROCK inhibitor. 
	NOTE: From this point on, the cells will be grown under 37 &#176;C/5% CO2.</li>
	<li>Two days later, change medium for wells containing undifferentiated colonies. Supplement the medium with 10 &#181;M ROCK inhibitor and 0.5 &#181;g/mL puromycin. 
	NOTE: In some wells, the cells might have differentiated and need to be discarded.</li>
	<li>Use P2 pipette tips to scrape off the colonies gently. Transfer the cell suspension derived from one colony into 2 new wells on separate 96-well plates, and use one for genotyping and one for maintaining. 
	NOTE: It is important to carefully maintain the colonies and keep the spontaneous differentiation level to the minimum.</li>
	<li>Take the plate containing cells for genotyping out once the confluency in most wells reached 50% or above. Dump the spent medium and then wash the cells once with DPBS.</li>
	<li>Lyse the cells in well using Bradley lysis buffer and isolate genomic DNA from each well in the plate following associated protocol (https://mcmanuslab.ucsf.edu/protocol/dna-isolation-es-cells-96-well-plate).</li>
	<li>Use a three-primer junction PCR method to genotype both left and right homology arms independently10. 
	NOTE: A scheme showing the PCR method is presented in Figure 2.</li>
	<li>Send the junction PCR products for both homology arms from 4-5 colonies for Sanger sequencing and get the sequences. 
	NOTE: The sequencing result of 2 established colonies is shown in Figure 3A.</li>
	<li>Keep colonies with the correct genotype and discard the rest.</li>
	<li>Expand the correct colonies under continuous puromycin selection and freeze them at the earliest possible passage.</li>
</ol>
4. Removing transposon from targeted human pluripotent stem cells
<ol>
	<li>Maintain one colony with correct genotype under 0.5 &#181;g/mL puromycin selection.</li>
	<li>Nucleofect 8 x 105 to 1 x 106 cells with 5 &#181;g hyperactive transposase (pCMV-hyPBase) as described above (Section 3, steps 3.4-3.18). Perform a GFP control nucleofection in parallel. 
	NOTE: Puromycin should be removed from the mTeSR1 medium immediately after nucleofecting these cells.</li>
	<li>Grow and screen for cells with the puro-deltaTK selection cassette removed following published procedures10. 
	NOTE: It is important to follow the original procedures carefully. FIAU, or 1-(2-deoxy-2-fluoro-&#946;-d-arabinofuranosyl)-5-iodouracil), was used as a thymidine analog for Herpes simplex virus-derived thymidine kinase (HSV-tk)-based negative selection.</li>
	<li>Sequence the modified region to confirm the removal of the transposon. 
	NOTE: The sequencing result of 3 established colonies is shown in Figure 3B.</li>
	<li>Keep colonies with the correct genotype and expand for further usage.</li>
	<li>Perform characterization of pluripotency markers in the chosen colonies before using them for further analysis. 
	NOTE: The characterization of two established colonies is shown in Figure 4.</li>
</ol>

<ol>
	<li>Rashidi, 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=3D+human+liver+tissue+from+pluripotent+stem+cells+displays+stable+phenotype+in+vitro+and+supports+compromised+liver+function+in+vivo.">3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.</a> Archives of Toxicology. 92 (10), 3117-3129 (2018).</li><li>Hockemeyer, 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=Efficient+targeting+of+expressed+and+silent+genes+in+human+ESCs+and+iPSCs+using+zinc-finger+nucleases.">Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.</a> Nature Biotechnology. 27 (9), 851-857 (2009).</li><li>Porteus, M. H., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+nucleases+stimulate+gene+targeting+in+human+cells.">Chimeric nucleases stimulate gene targeting in human cells.</a> Science. 300 (5620), 763 (2003).</li><li>Watanabe, K., 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+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</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, 816-822 (2012).</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 (6), 1380-1389 (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 (6121), 819-824 (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 (6121), 823-826 (2013).</li><li>Yusa, K., 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=Targeted+gene+correction+of+&#945;1-antitrypsin+deficiency+in+induced+pluripotent+stem+cells.">Targeted gene correction of &#945;1-antitrypsin deficiency in induced pluripotent stem cells.</a> Nature. 478 (7369), 391-394 (2011).</li><li>Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seamless+genome+editing+in+human+pluripotent+stem+cells+using+custom+endonuclease-based+gene+targeting+and+the+piggyBac+transposon.">Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon.</a> Nature Protocols. 8 (10), 2061-2078 (2013).</li><li>Wang, 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=Multiomics+Analyses+of+HNF4&#945;+Protein+Domain+Function+during+Human+Pluripotent+Stem+Cell+Differentiation.">Multiomics Analyses of HNF4&#945; Protein Domain Function during Human Pluripotent Stem Cell Differentiation.</a> iScience. 16, 206-217 (2019).</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. <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+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Wang, 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=Defined+and+Scalable+Generation+of+Hepatocyte-like+Cells+from+Human+Pluripotent+Stem+Cells.">Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells.</a> Journal of Visualized Experiments. 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D.C.H is a co-founder, shareholder, and director of Stemnovate Limited.

The protocol described herein can be used to introduce&#160;predetermined point mutations into an endogenous locus in hPSCs. The combination of a piggyBac-based targeting vector and paired CRISPR/Cas9n expression plasmids proved to be reliable and efficient11. Following HNF4&#945;&#160;gene editing, 12 out of 43 analyzed clones were correctly targeted as determined by junction PCR screening. Specifically, the biallelic targeting efficiency was about 21% (9/43) and the monoallelic targeting efficiency was around 7% (3/43).
Following targeting experiments, it is crucial to maintain&#160;puromycin selection to keep the targeted locus accessible to the piggyBac transposase during the first round of screening. This will minimize the silencing of the PGK promoter-driven puro-deltaTK selection cassette and&#160;reduce the background&#160;in the second round of screening10. A recent report showed that CAG promoter is more&#160;resistant to silencing than PGK promoter in hPSCs14. Changing the promoter for the selection cassette could therefore&#160;improve this system&#160;in the future. It is also important to compare the number of resistant colonies from hyPBase- and GFP-transfected cells. Upon successful removal of the transposon, there should be more surviving colonies from the hyPBase- than GFP-transfected cells. In addition to PCR analysis of transposon excision, direct sequencing of the modified region is required to confirm the excision fidelity.
Based on Yusa&#39;s piggyBac transposon-based targeting vector strategy9,10, the procedures detailed above focused on improving hPSCs nucleofection and single cell cloning efficiency. Cell seeding density was optimized to reduce the likelihood of heterogeneous colony formation. We also employed 10-12 days culture under 10% CO2 to enhance colony production for genotype screening. In our experience, around 20 colonies could be obtained from each 96-well plate. Although this step might take more time than other methods, it is reliable and highly efficient.
Based on the need, targeting vectors can be designed to introduce or correct point mutations, to create reporter cell lines, as well as knockout gene expression. In conclusion, the combination of the&#160;CRISPR/Cas9(n) system and a targeting vector is an efficient way to deliver different types of genetically modified hPSCs.

Human pluripotent stem cells (hPSCs) represent an unlimited source of somatic cells for research or the clinic1. Gene targeting in hPSCs offers a powerful method to study gene function during cell specification and to understand mechanisms of disease. Although efficient genome editing methods exist, modifying genes in hPSCs remains technically challenging. Standard gene targeting by homologous recombination in hPSCs occurs at low frequency or is even undetectable at some genes2, and DNA double-strand break (DSB) stimulated gene targeting (termed gene editing) is therefore necessary in these cells2,3. Additionally, transfection of hPSCs and subsequent single-cell cloning is not very efficient, even though single cell-associated apoptosis can be reduced through the use of the Rho-associated protein kinase (ROCK) inhibitor4. Finally, the potential on-target and off-target mutations at the gene of interest can also be problematic. Hence, a reliable protocol is essential to make tailored genetic changes in hPSCs.
The RNA-guided CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated protein 9 (Cas9) technology is now a well-established tool in gene editing. Transcribed CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA) form the single guide RNA (sgRNA), which complexes with Cas9 protein allowing gene specific cleavage5. The CRISPR/Cas9 system is programmable, by changing a 20-bp guide sequence of the sgRNA, meaning almost any locus that fulfils the protospacer-adjacent motif (PAM) requirement can be edited. However, the wild type Cas9 can tolerate some mismatches between its guide sequence and a DNA target, which might cause unwanted off-target effects. To improve its specificity, a nickase mutant (Cas9n) has been developed. A Cas9n containing D10A or N863A mutation only possesses one functional nuclease domain and as a result can only nick DNA on one strand. A pair of Cas9n appropriately spaced and oriented can effectively induce DNA DSB and the off-target effects are dramatically reduced6. To ensure efficient Cas9n modification, it has been shown that two Cas9n-sgRNA should ideally be placed with a -4 to 20 bp offset and always create a 5&#39; overhang6.
Cas9(n)-sgRNA induced DSB can be utilized for genome editing in hPSCs7,8. It can create gene knockout via non-homologous end joining repair, or introduce gene modification through homology-directed repair (HDR) if a donor DNA template is present. The protocol described here uses a piggyBac transposon-based donor plasmid (or targeting vector) for HDR, in which a drug resistance marker is flanked by the transposon inverted repeats. The advantage of this approach includes efficient screening and seamless gene editing as firstly demonstrated by Yusa et al.9,10. The drug selection cassette allows enrichment of cells with the integrated vector, which are subsequently screened by junction PCR to identify those derived by HDR. In addition, the drug selection cassette can be positioned to replace the target sequences for Cas9(n) cleavage, so no further DNA breakage occurs after HDR, thus eliminating &#39;on&#39; target mutations resulting from Cas9(n) re-cleavage. Furthermore, by exploiting the precise excision catalyzed by piggyBac transposase, the selection marker is then excised from the genome without leaving a scar. Only the original endogenous TTAA sequence for piggyBac insertion remains after the removal of the drug selection marker9. Even if the TTAA sites have to be created by introducing substitutions, the chances of disturbing regulatory elements are reduced compared to other methods10.
Here we describe detailed procedures for implementing seamless genome editing in hPSCs. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we introduced two predetermined point mutations into the hepatocyte nuclear factor 4 alpha (HNF4&#945;) gene11. This approach is reliable and efficient, and has permitted in-depth analyses of the important roles played by HNF4&#945; in endoderm and hepatocyte specification from human pluripotent stem cells11.

<table><tbody><tr><td>Anti-Human SSEA4 PE</td><td>eBioscience</td><td>12-8843-42</td><td></td></tr><tr><td>Anti-Human TRA-1-60 PE</td><td>eBioscience</td><td>11-0159-42</td><td></td></tr><tr><td>DPBS</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>DPBS with calcium and magnesium</td><td>Thermo Fisher Scientific</td><td>14040133</td><td></td></tr><tr><td>FIAU</td><td>Moravek</td><td>M251</td><td></td></tr><tr><td>Gentle cell dissociation reagent</td><td>Stem Cell Technologies</td><td>07174</td><td></td></tr><tr><td>H9 human embryonic stem cells</td><td>WiCell</td><td>WA09</td><td></td></tr><tr><td>Human NANOG antibody</td><td>R&amp;amp;D systems</td><td>AF1997</td><td></td></tr><tr><td>Human OCT4 antibody</td><td>Abcam</td><td>AB19857</td><td></td></tr><tr><td>Human stem cell Nucleofector kit 1</td><td>Lonza</td><td>VPH-5012</td><td></td></tr><tr><td>Mouse IgG3 isotype control PE</td><td>eBioscience</td><td>12-4742-41</td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem Cell Technologies</td><td>85850</td><td></td></tr><tr><td>Nucleofector 2b device</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>pCMV-hyPBase</td><td>Wellcome Trust Sanger Institute</td><td>N/A</td><td></td></tr><tr><td>pMCS-AAT_PBPGKpuroTK</td><td>Wellcome Trust Sanger Institute</td><td>N/A</td><td></td></tr><tr><td>pSpCas9n(BB)-2A-Puro (PX462)</td><td>Addgene</td><td>PX462</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Thermo Fisher Scientific</td><td>A11138-03</td><td></td></tr><tr><td>QIAprep spin maxiprep kit</td><td>Qiagen</td><td>12162</td><td></td></tr><tr><td>QIAprep spin miniprep kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Recombinant laminin 521</td><td>BioLamina</td><td>LN521</td><td></td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Y-27632(Dihydrochloride)</td><td>Millipore</td><td>SCM075</td><td></td></tr></tbody></table>

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.

Targeting vector-based knock-in strategy 
The hepatocyte nuclear factor 4 alpha (HNF4&#945;) gene was chosen for targeted genome editing to introduce two point mutations into exon 8. A pair of Cas9n-sgRNA with 11 bp offset was designed close to the site to be modified. The piggyBac-based targeting vector worked as the homology-directed repair template for introducing the desired point mutations. A 967 bp 5&#39;-HA and a 1142 bp 3&#39;-HA incorporating synonymous nucleotide substitutions or the desired point mutations were amplified and cloned into the final targeting vector. The piggyBac insertion site was 16 bp and 22 bp away from the two desired point mutations. Colonies containing the puro-deltaTK selection cassette were selected with puromycin in the first round. Once the selection cassette was removed by transposase excision in the second round, the targeted site was modified seamlessly with only the desired point mutations incorporated in the gene (Figure 1).
Genetic editing in human pluripotent stem cells 
To screen for correctly targeted cells, a three primer-based PCR method was used for genotyping (Figure 2). Sanger sequencing was performed to confirm the PCR results (Figure 3A). Post removal of the selection cassette, the modified region was sequenced again to confirm the correct introduction of desired point mutations (Figure 3B).
Colony establishment and characterization of edited human pluripotent stem cells 
Colonies with the correct genotype were selected and expanded as needed. The established colonies need to be characterized before being used for further analysis. The edited cells possess the same morphology as the parental cells (Figure 4A). They also express representative human pluripotent stem cell markers, including transcription factors NANOG and OCT4 (Figure 4B), as well as cell surface markers SSEA4 and TRA-1-60 (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61152/61152fig1.jpg" /> 
Figure 1: Targeting vector-based knock-in strategy11. A pair of Cas9n-sgRNA expression plasmids were used to induce DNA double-strand break in exon 8 of the HNF4&#945; gene. A targeting vector with a selection cassette was used to introduce predetermined point mutations located 16 bp and 22 bp away. This selection cassette was contained within the piggyBac transposon, consisting of a positive-negative selection marker (puro-deltaTK) driven by a constitutively active promoter (PGK). Via homology-directed repair pathway, the targeted cells incorporated the selection cassette. Transposon excision mediated by transposase results in a seamless modification with only the point mutations present. Red cross indicates the location of desired point mutations. HA = homology arm; PB = piggyBac; PM = point mutation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61152/61152fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61152/61152fig2.jpg" /> 
Figure 2: Three primer-based PCR method. To screen gene targeted cells, PCR-based genotyping was used. Three primers, LA-F1, -R1 and -R2 were used to amplify the left homology arm region. Independently, RA-F1, -F2 and -R1 were used to amplify the right homology arm region. Based on the gel electrophoresis result, non-targeted cells, and heterozygous and homozygous cells, were distinguished from each other. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61152/61152fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61152/61152fig3.jpg" /> 
Figure 3: Sequencing results of genetically modified cells11. PCR products from two clones were sequenced and confirmed the correct insertion of the selection cassette at the targeted locus (A). 5&#39; and 3&#39; piggyBac inverted terminal repeats (ITR) were flanked by the TTAA direct repeats. Three clones were sequenced post transposon excision (B). A pair of Cas9n-sgRNA with 11 bp offset was used to introduce DNA double-strand break. Two predetermined point mutations (A to G and A to C) were introduced into the gene. One synonymous mutation was introduced to mutate the protospacer-adjacent motif (PAM), and another one to create the TTAA site necessary for piggyBac excision. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61152/61152fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61152/61152fig4.jpg" /> 
Figure 4: Characterization of edited human pluripotent stem cells. Morphology of two edited cell lines and the parental cells (A), scale bar = 100 &#181;m. The expression of pluripotent stem cell markers NANOG and OCT4 examined by immunostaining (B, scale bar = 50 &#181;m), in addition to SSEA4 and TRA-1-60 by flow cytometry (C) in two modified cell lines and the parental cells. IgG was used as a negative control. DAPI was used to stain the nucleus. The percentages were calculated as the average of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61152/61152fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing at JoVE.com

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

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 ...

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4.0K Views.  University of Edinburgh. 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 (HNF4α) locus in human embryonic stem cells (hESCs).

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

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Developmental Biology

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 ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

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 ...

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 ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Medicine

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical ...

Biology

Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

Source: Blair, J. D., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/53583">Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.</a> J. Vis. Exp. (2016).
This video demonstrates a technique of zinc-finger nuclease-based genome editing in human pluripotent stem cells (hPSCs). As hPSCs can potentially be differentiated into various cell types, this technology is helpful for in vitro pathological and pharmacological studies in a patient-specific manner.

Source: Blair, J. D., et al. Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation. J. Vis. Exp. (2016).
This ...

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TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs

Source: Cerbini, T. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52504">Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor.</a> J. Vis. Exp. (2015)
This video describes a precise genome editing technique in human induced pluripotent stem cells, or hiPSCs, using TALENs to create double-stranded breaks at a targeted locus, inducing homology-directed repair for fluorescence protein gene integration.

Source: Cerbini, T. et al. Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the ...

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

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Chapters in this video

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

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

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

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

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

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

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

TALEN-Mediated Targeted Gene Integration: Using Engineered Sequence-Specific Nucleases for the Precise Insertion of Fluorescent Protein Gene into hiPSCs