We thank Muthugapatti Kandasamy at the University of Georgia (UGA) Biomedical Microscopy Core for technical assistance and Jose-Juan Lopez-Rubio for sharing the pUF1-Cas9 and pL6 plasmids. This work was supported by ARCS Foundation awards to D.W.C. and to H.M.K., UGA startup funds to V.M., grants from the March of Dimes Foundation (Basil O&#39;Connor Starter Scholar Research Award) to V.M., and US National Institutes of Health grants (R00AI099156 and R01AI130139) to V.M. and (T32AI060546) to H.M.K.

Continuous culture of P. falciparum requires the use of human RBCs, and we utilized commercially purchased units of blood that were stripped of all identifiers and anonymized. The Institutional Review Board and the Office of Biosafety at the University of Georgia reviewed our protocols and approved all protocols used in our lab.
1. Choosing a gRNA Sequence
<ol>
	<li>Go to CHOPCHOP (http://chopchop.cbu.uib.no/) and select &#39;Fasta Target&#39;. Under &#39;Target&#39;</e...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2017).</li><li>Miller, L. H., Baruch, D. I., Marsh, K., Doumbo, O. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+heat+shock+protein+110+stabilizes+the+asparagine+repeat-rich+parasite+proteome+during+malarial+fevers.">Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers.</a> Nature Communications. 3, 1310 (2012).</li><li>Muralidharan, V., Oksman, A., Iwamoto, M., Wandless, T. J., Goldberg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asparagine+repeat+function+in+a+Plasmodium+falciparum+protein+assessed+via+a+regulatable+fluorescent+affinity+tag.">Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag.</a> Proceedings of the National Academy of Sciences the USA. 108 (11), 4411-4416 (2011).</li><li>Beck, J. R., Muralidharan, V., Oksman, A., Goldberg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEX+component+HSP101+mediates+export+of+diverse+malaria+effectors+into+host+erythrocytes.">PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes.</a> Nature. 511 (7511), 592-595 (2014).</li><li>Ghorbal, 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=Genome+editing+in+the+human+malaria+parasite+Plasmodium+falciparum+using+the+CRISPR-Cas9+system.">Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system.</a> Nature Biotechnology. 32 (8), 819-821 (2014).</li><li>Wagner, J. C., Platt, R. J., Goldfless, S. J., Zhang, F., Niles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+CRISPR-Cas9-mediated+genome+editing+in+Plasmodium+falciparum.">Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum.</a> Nature Methods. 11 (9), 915-918 (2014).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing.+The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">Genome editing. The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li><li>Wang, H., La Russa, M., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+Genome+Editing+and+Beyond.">CRISPR/Cas9 in Genome Editing and Beyond.</a> Annual Review of Biochemistry. 85, 227-264 (2016).</li><li>Kirkman, L. A., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+and+the+generation+of+diversity+in+malaria+parasites.">Antigenic variation and the generation of diversity in malaria parasites.</a> Current Opinion in Microbiology. 15 (4), 456-462 (2012).</li><li>Lee, A. H., Symington, L. S., Fidock, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Repair+Mechanisms+and+Their+Biological+Roles+in+the+Malaria+Parasite+Plasmodium+falciparum.">DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum.</a> Microbiology and Molecular Biology Reviews. 78 (3), 469-486 (2014).</li><li>Cobb, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Exported+Chaperone+PfHsp70x+Is+Dispensable+for+the+Plasmodium+falciparum+Intraerythrocytic+Life+Cycle.">The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.</a> mSphere. 2 (5), (2017).</li><li>Prommana, 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=Inducible+knockdown+of+Plasmodium+gene+expression+using+the+glmS+ribozyme.">Inducible knockdown of Plasmodium gene expression using the glmS ribozyme.</a> Public Library of Science One. 8 (8), e73783 (2013).</li><li>Ganesan, S. M., Falla, A., Goldfless, S. J., Nasamu, A. S., Niles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+RNA-protein+modules+integrated+with+native+translation+mechanisms+to+control+gene+expression+in+malaria+parasites.">Synthetic RNA-protein modules integrated with native translation mechanisms to control gene expression in malaria parasites.</a> Nature Communications. 7, 10727 (2016).</li><li>Li, M. Z., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+homologous+recombination+in+vitro+to+generate+recombinant+DNA+via+SLIC.">Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.</a> Nature Methods. 4 (3), 251-256 (2007).</li><li>Drew, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+food+vacuole+plasmepsins+are+activated+by+falcipains.">Plasmodium food vacuole plasmepsins are activated by falcipains.</a> Journal of Biological Chemistry. 283 (19), 12870-12876 (2008).</li><li>Wu, Y., Sifri, C. D., Lei, H. -. H., Su, X. -. Z., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+Plasmodium+falciparum+within+human+red+blood+cells.">Transfection of Plasmodium falciparum within human red blood cells.</a> Proceedings of the National Academy of Sciences USA. 92, 973-977 (1995).</li><li>Janse, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transfection+of+Plasmodium+berghei+facilitates+novel+selection+procedures.">High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.</a> Molecular and Biochemical Parasitology. 145 (1), 60-70 (2006).</li><li>Counihan, N. 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=Plasmodium+falciparum+parasites+deploy+RhopH2+into+the+host+erythrocyte+to+obtain+nutrients,+grow+and+replicate.">Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate.</a> eLife. 6, (2017).</li><li>Klemba, M., Beatty, W., Gluzman, I., Goldberg, D. 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B., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v2:+a+web+tool+for+the+next+generation+of+CRISPR+genome+engineering.">CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.</a> Nucleic Acids Resesarch. 44 (W1), W272-W276 (2016).</li><li>Peng, D., Tarleton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EuPaGDT:+a+web+tool+tailored+to+design+CRISPR+guide+RNAs+for+eukaryotic+pathogens.">EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens.</a> Microbial Genomes. 1 (4), e000033 (2015).</li><li>Shen, B., Brown, K. M., Lee, T. D., Sibley, L. 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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=CRISPR-Cas9-modified+pfmdr1+protects+Plasmodium+falciparum+asexual+blood+stages+and+gametocytes+against+a+class+of+piperazine-containing+compounds+but+potentiates+artemisinin-based+combination+therapy+partner+drugs.">CRISPR-Cas9-modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs.</a> Molecular Microbiology. 101 (3), 381-393 (2016).</li><li>Lim, M. 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=UDP-galactose+and+acetyl-CoA+transporters+as+Plasmodium+multidrug+resistance+genes.">UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.</a> Nature Microbiology. , (2016).</li><li>Adjalley, S. 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=Quantitative+assessment+of+Plasmodium+falciparum+sexual+development+reveals+potent+transmission+blocking+activity+by+methylene+blue.">Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission blocking activity by methylene blue.</a> Proceedings of the National Academy of Sciences USA. 108 (47), E1214-E1223 (2011).</li><li>Sidik, S. 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The implementation of CRISPR/Cas9 in P. falciparum has both increased the efficiency of and decreased the amount of time needed for modifying the parasite&#39;s genome, compared to previous methods of genetic manipulation. This comprehensive protocol outlines the steps necessary for generating conditional mutants using CRISPR/Cas9 in P. falciparum. While the method here is geared specifically for the generation of HA-glmS mutants, this strategy can be adapted for a variety of needs, including t...

Malaria is a devastating disease caused by protozoan parasites of the genus Plasmodium. P. falciparum, the most deadly human malaria parasite, causes approximately 445,000 deaths per year, mostly in children under the age of five1. Plasmodium parasites have an intricate life cycle involving a mosquito vector and a vertebrate host. Humans first become infected when an infected mosquito takes a blood meal. Then, the parasite invades the liver where it grows, develops, and divides for approximately one week.After this process, the parasites are released into the bloodstream, where they undergo asexual replication in red blood cells (RBCs). Growth of the parasites within the RBCs are directly responsible for the clinical symptoms associated with malaria2.
Until recently, production of transgenic P. falciparum was a laborious process, involving several rounds of drug selection that took many months and had a high failure rate. This time-consuming procedure relieson the generation of random DNA breaks in the region of interest and the endogenous ability of the parasite to mend its genome though homologous repair3,4,5,6. Recently, Clustered Regularly Interspaced Palindromic Repeat/Cas9 (CRISPR/Cas9) genome editing has been successfully utilized in P. falciparum7,8. The introduction of this new technology in malaria research has been critical for advancing understanding of the biology of these deadly Plasmodium parasites. CRISPR/Cas9 allows for specific targeting of genes through guide RNAs (gRNAs) that are homologous to the gene of interest. The gRNA/Cas9 complex recognizes the gene through the gRNA, and Cas9 then introduces a double-strand break, forcing the initiation of repair mechanisms in the organism9,10. Because P. falciparum lacks the machinery to repair DNA breaks through non-homologous end joining, it utilizes homologous recombination mechanisms and integrates transfected homologous DNA templates to repair the Cas9/gRNA-induced double-strand break11,12.
Here, we present a protocol for the generation of conditional knockdown mutants in P. falciparum using CRISPR/Cas9 genome editing. The protocol demonstrates usage of the glmS ribozyme to conditionally knockdown protein levels of PfHsp70x (PF3D7_0831700), a chaperone exported by P. falciparum into host RBCs13,14. The glmS ribozyme is activated by treatment with glucosamine (which is converted to glucosamine-6-phosphate in cells) to cleave its associated mRNA, leading to a reduction in the protein14. This protocol can be easily adapted to utilize other conditional knockdown tools, such as destabilization domains or RNA aptamers4,5,15. Our protocol details the generation of a repair plasmid consisting of a hemagglutinin (HA) tag and glmS ribozyme coding sequence flanked by sequences that are homologous to the PfHsp70x open reading frame (ORF) and 3&#39;-UTR. We also describe the generation of a second plasmid to drive expression of the gRNA. These two plasmids, along with a third that drives expression of Cas9, are transfected into RBCs and used to modify the genome of P. falciparum parasites. Finally, we describe a polymerase chain reaction (PCR)-based technique to verify integration of the tag and glmS ribozyme. This protocol is highly adaptable for the modification or complete knockout of any P. falciparum genes, enhancing our ability to generate new insights into the biology of the malaria parasite.

<table>
	<tbody>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>Bio-Rad</td>
			<td>165-2086</td>
			<td>We buy the ones that are individually wrapped</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>DSM1</td>
			<td>Gift from Akhil Vaidya lab</td>
			<td>Ganesan et al. Mol. Biochem. Parasitol. 2011 177:29-34</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 6 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP 100mm Tissue Culture Dishes (12 mL Plate)</td>
			<td>MIDSCI</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 96 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92096</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 24 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92024</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 2</td>
			<td>New England Biolabs</td>
			<td>#B7002S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 2.1</td>
			<td>New England Biolabs</td>
			<td>#B7202S</td>
			<td></td>
		</tr>
		<tr>
			<td>BtgZI</td>
			<td>New England Biolabs</td>
			<td>#R0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacII</td>
			<td>New England Biolabs</td>
			<td>#R0157L</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII-HF</td>
			<td>New England Biolabs</td>
			<td>#R3104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Afe1</td>
			<td>New England Biolabs</td>
			<td>#R0652S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nhe1-HF</td>
			<td>New England Biolabs</td>
			<td>#R3131L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>#M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Steritop bottle top filter unit</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
			<td>You can use any size that fits your needs</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C7902-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>pMK-U6</td>
			<td>Generated by the Muralidharan Lab</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>pHA-glmS</td>
			<td>Generated by the Muralidharan Lab</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>pUF1-Cas9</td>
			<td>Gift from the Jose-Juan Lopez-Rubio Lab</td>
			<td>Ghorbal et al. Nature Biotech 2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>H9636-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PL6-eGFP BSD</td>
			<td>Generated by the Muralidharan Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Puf1-cas9 eGFP gRNA</td>
			<td>Generated by the Muralidharan Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax I</td>
			<td>Life Technologies</td>
			<td>N/A</td>
			<td>You will want to try a few batches to find out what the parasites will grow in best</td>
		</tr>
		<tr>
			<td>Human Red Blood Cells</td>
			<td>Interstate Blood Bank, Inc</td>
			<td>Email or call them directly for ordering</td>
			<td>We typically use O+ blood</td>
		</tr>
		<tr>
			<td>3D7 parasite line</td>
			<td>Available upon request</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>Fisher</td>
			<td>BP1426-2</td>
			<td>You can make your own, it is not necessary to use exactly this</td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Fisher</td>
			<td>BP1760-25</td>
			<td>We make a 1000X stock at 100mg/ml in water and store in the -20C</td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Clonetech</td>
			<td>R050A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EF1alpha</td>
			<td>Dr. Daniel Goldberg&#39;s Lab</td>
			<td>Washington University in St. Louis</td>
			<td>You can use your preferred loading control for western blots. This is just the one we use in our laboratory</td>
		</tr>
		<tr>
			<td>Rat Anti-HA Clone 3F10, monoclonal</td>
			<td>Made by Roche, sold by Sigma</td>
			<td>11867423001</td>
			<td>You can use your preferred anti-HA antibody</td>
		</tr>
		<tr>
			<td>0.6 mL tubes</td>
			<td>Fisher</td>
			<td>AB0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher HealthCare* PROTOCOL* Hema 3* Manual Staining System (Fixative+Solution I and II)</td>
			<td>Fisher</td>
			<td>22-122-911</td>
			<td>You can also use giemsa stain</td>
		</tr>
		<tr>
			<td>Fisherfines Premium Frosted Microscope Slides - Size: 3 x 1 in.</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr/cas9 gene editing to make conditional mutants of human malaria parasite p. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

A schematic of the plasmids used in this method as well as an example of a shield mutation are shown in Figure 1. As an example of how to identify mutant parasites after transfection, results from PCRs for checking integration of the HA-glmS construct are shown in Figure 2. A representative image of a cloning plate is shown in Figure 3 to demonstrate the color change of the medium in the pre...

Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum at JoVE.com

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

This method can help answer key questions in the biology of the human malaria parasite, P.Falciparum, by helping to uncover protein function through conditional knockdown. The main advantage of this technique is that it allows for relatively easy generation of conditional mutants as compared to previous methods. To begin, go to Chop Chop and select Fasta Target'Under Target, paste the 200 base pairs from the three prime end of the open reading frame of a gene and 200 base pairs from the start of the gene's three prime UTR.Under In'select P.Falciparum'and select CRISPR/Cas9'under Using'Next, click Find Target Sites'Select a gRNA sequence from the options presented, giving preference to the most efficient gRNA that is closest to the site of modification and that has the fewest off-target sites. Purchase the gRNA sequence and its reverse complement as polyacrylamide gel electrophoresis purified oligos. The gRNA sequence used to target PfH-sp70x can be found in the text protocol.Add 40 micrograms each of pMK-U6, pUF1-Cas9, and pHA-glmS DNA into a sterile 1.5 millimeter centrifuge tube. Add one-tenth of the volume of DNA of three molar sodium acetate in water to the tube and mix it well using a vortex. Then, add 2.5 times the volume of 100 percent ethanol to the tube and mix it well using a vortex for at least 30 seconds.Place the tube on ice or at minus 20 degrees Celsius for 30 minutes. Then, centrifuge the tube at 18, 300 times G for 30 minutes at four degrees Celsius. Following centrifugation, carefully remove the supernatant from the tube without disturbing the pellet.Add three times the volume of 70 percent ethanol to the tube and mix it briefly using a vortex. Centrifuge the tube at 18, 300 times G for 30 minutes at four degrees Celsius. Again, carefully remove the supernatant from the tube without disturbing the pellet.Leave the tube open and allow the pellet to air dry for 15 minutes. Store the precipitated DNA at minus 20 degrees Celsius until it is needed for transfection. To transfect RBCs, prepare 1X cytomix buffer as well as incomplete and complete RPMI as described in the text protocol.Filter sterilize the buffer using a 0.22 micron filter. Add 380 microliters of the 1X cytomix to the DNA and vortex to dissolve. Allow the DNA to dissolve in the 1X cytomix for 10 minutes, vortexing every three minutes for 10 seconds.In a sterile, 15 milliliter conical tube, combine 300 microliters of isolated human RBCs in the incomplete RPMI with four milliliters of 1X cytomix. Centrifuge the RBCs at 870 times G for three minutes. Then remove the supernatant from the RBC pellet.Re-suspend the RBC pellet with the DNA-cytomix mixture and transfer it to a 0.2 centimeter electroporation cuvette. Porate the RBCs using the conditions listed in the text protocol. Following electroporation, transfer the contents from the cuvette to a 15 milliliter conical tube containing five milliliters of complete RPMI.Centrifuge the tube at 870 times G for three minutes at 20 degrees Celsius. Then, decant the supernatant. Now, re-suspend the pellet in four milliliters of complete RPMI and transfer to one well in a six-well tissue culture plate.Add 400 microliters of a high schizont culture to the transferred RBCs. Maintain all the cultures at 37 degrees Celsius under three percent oxygen, three percent CO2, and 94 percent nitrogen. The next day, wash the culture with four milliliters of complete RPMI.Centrifuge the culture at 870 times G for three minutes. Aspirate the supernatant. Re-suspend the culture in four milliliters of complete RPMI.48 hours later, wash the culture with four milliliters of complete RPMI. Then, re-suspend the culture in complete RPMI containing one micromolar DSM1 to select for the Cas9 plasmid. After this point, replace the culture medium with fresh complete RPMI plus one micromolar DSM1 every 48 hours.Continue washing the cultures each day with complete RPMI until parasites are no longer visible by blood smear. To make a blood smear, pipette 150 microliters of culture into a 0.6 milliliter centrifuge tube. After pelleting the cells by centrifugation at 1, 700 times G for 30 seconds, aspirate off the supernatant.Use a pipette to transfer the pelleted cells to a glass slide. Using a second glass slide held at a 45 degree angle to the first slide, smear the blood droplet. Stain the slide using a commercially available staining kit according to the manufacturer's protocol.View the parasites using 100 times oil-immersion objective. Beginning five days post-transfection, remove two milliliters of the culture with RBCs re-suspended in the culture medium. Add back two milliliters of fresh medium and blood at two percent hematocrit.Add fresh blood in this manner once a week until parasite reappear, as determined by thin blood smear. Perform serial dilutions of the parasite culture to achieve a final concentration of 0.5 parasites per 200 microliters. Add 200 microliters of the diluted culture to the wells of a 96-well tissue culture plate.Maintain the cloning plate until parasites are detectable in the wells. Every 48 hours, replace the medium in the 96-well plate with fresh medium. Once a week, starting five days after beginning the cloning plate, remove 100 microliters from each well and add back 100 microliters of fresh medium plus blood.To identify any wells containing parasites, place the 96-well plate at a 45 degree angle for approximately 20 minutes, allowing the blood to settle at an angle within the plate. Then, place the 96-well plate on a light box. Notice that the wells containing parasites contain media that is yellow in color, compared to the pink media of parasite-free wells, due to acidification of the medium by the parasites.Using a serological pipette, move the contents of the parasite-containing wells to a 24-well tissue culture plate to allow expansion of the parasitemia. Using PCR analysis, check these clonal parasite lines for correct integration. The results of an immunofluorescence assay are shown here, demonstrating the successfully HA tagging of PfH-sp70x.PfH-sp70x glmS parasites were fixed and stained with DAPI as a nucleus marker, as well as antibodies to HA.Membrane-associated histidine rich protein one, a marker of protein export to the host RBC. A Western Blot analysis of PfH-sp70x protein level after treatment with glucosamine is shown here. As expected, the protein level decreases during the duration of treatment.This technique paves the way for researchers in the field of parasitology to investigate fundamental questions in the biology of the malaria parasite. It's important to remember that P.Falciparum is a blood-borne pathogen and appropriate personal protective equipment should be worn while performing this procedure.

crisprcas9-gene-editing-to-make-conditional-mutants-human-malaria

Research

JoVE Journal

Genetics

9.8K 조회수.  University of Georgia. 이 방법은 조건부 녹다운을 통해 단백질 기능을 발견하는 것을 도움으로써 인간 말라리아 기생충, P.Falciparum의 생물학에 있는 중요한 질문에 대답하는 것을 도울 수 있습니다. 이 기술의 주요 장점은 이전 방법에 비해 조건부 돌연변이의 비교적 쉬운 생성을 허용한다는 것입니다. 시작하려면, 잘라 잘라 내고 Fasta Target'Under Target'Under Target을 선택하고 유전자의 열린 판독 프레?...