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. (121), 1-8 (2017).</li><li>Eggenschwiler, R., 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=Improved+bi-allelic+modification+of+a+transcriptionally+silent+locus+in+patient-derived+iPSC+by+Cas9+nickase.">Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.</a> Scientific Reports. 6, 1-14 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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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Nanyang Technological University

Research

JoVE Journal

Genetics

3.9K 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

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

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 ...

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

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

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

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. ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

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

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 ...

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

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

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

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

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

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

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

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...