We would like to thank L. David Sibley and Asis Khan for their contribution to the original publication on which these protocols are based. This work was funded by National Institutes of Health grant AI108721.

The authors declare that they have no competing financial interests.

These protocols present several methods that when combined allow for the identification of a drug resistance gene in T. gondii. Two in particular were integral to the project, the relatively seasoned method of QTL mapping and the recently developed method of CRISPR/Cas9 gene editing. Lander and Botstein published their influential paper in 1989 demonstrating QTL mapping which correlates genetic loci with phenotypes 36. More recently in 2012, Dounda and Charpentier described the CRISPR/Cas9 editing system in Streptococcus pyogenes 22 that was quickly adapted as a genetic tool in many different models, including T. gondii13,17. Both methods were useful here, where QTL mapping defined the locus containing the drug resistance mutation that was ultimately identified using WGS based SNP detection, and CRISPR/Cas9 editing provided the means to confirm SNR1 is the sinefungin drug resistance gene.
The ME49-FUDRr X VAND-SNFr cross 8 was originally developed to interrogate a virulence phenotype 19, but the parental strains also happened to have an additional phenotypic difference for which the causal gene was not known, sinefungin resistance in the VAND parent. This highlights one benefit of crosses in that they can be repurposed when additional phenotypic differences in the parents are observed. This was the case for another Toxoplasma cross, type 2 x type 3, where several genes involved in virulence were found by mapping multiple phenotypes 37,38,39,40. To date, four different T. gondii crosses have been described and used to map genes responsible for phenotypes 8,37,41,42, all of which have the potential to be reused to map new phenotypes for which the genetic basis is unknown. Along these lines, the genomes for 62 T. gondii strains representing the known global genetic diversity have been sequenced 43. New crosses could be made from this pool for strains that differ in interesting phenotypes. Having extolled the advantages of QTL mapping, it needs to be said that generating a cross isn&#39;t a minor undertaking. There are other methods that can be used to identify causal genes. One powerful technique uses chemical mutagenesis to create mutants that can be screened for phenotypes. To find the causal gene, mutants can either be complemented with cosmid libraries 44 or genome resequencing methods can be used to find the causal mutation 45. For more on this, see two JoVE articles by Coleman et al. and Walwyn et al. that outline these approaches 46,47.
Many of the steps leading to the identification of the causal SNFr mutation (Protocols 2 and 3) rely on computational methods conducted with software that is freely available for academic use. Detailed commands for each step are provided and when run with the proper files will allow the user to recreate the datasets necessary to find the causal SNFr SNP. Keep in mind that some of the syntax in the commands refer to filenames or directory structure ($PATH) that can be modified to the user preference. Although the commands given here certainly do not exhaust the ways one can analyze a cross with QTL analysis and WGS based SNP identification, they are comprehensive enough to repeat the experiment described in this article and should allow the user to become more familiar with how these approaches are utilized in a step by step fashion.
Although QTL mapping and WGS sequence based SNP detection were&#160;sufficient to identify a candidate SNFr gene, additional experiments are needed to confirm its role in drug resistance. This can be convincingly shown through gene disruption or knockout techniques, both of which can be achieved using CRISPR/Cas9 gene editing. Detailed methods for using CRISPR/Cas9 to either generate indel mutations or insert transgenic constructs into a target gene in T. gondii are given. The targeting specificity which CRISPR/Cas9 provides increases the efficiency of gene editing over traditional methods. Also, this increased efficiently has made it possible to use non-laboratory adapted strains for genetic studies which were previously difficult to modify 13. Even though in its infancy, CRISPR/Cas9 has already been used to make gene disruptions 2,13,17,18,48,49, tag genes 17, and make gene knockouts 16,19,50,51,52,53,54 in T. gondii, promising to be a useful tool for many other studies in the future.
The discovery that SNR1 inactivation leads to sinefungin resistance makes the SNR1 locus a promising site for transgene insertion or genetic complementation. To maximize the success of using CRISPR/Cas9 mediated gene targeting to direct the integration of transgene into the SNR1 locus, the following aspects should be considered during the experimental design. First, a selection marker is recommended to be included in the transgenic construct to increase the efficiency of strain construction. If a selection marker is included, both positive and negative selection can be used, resulting in almost 100% of the doubly selected parasites being transgenic with the GOI integrated at the SNR1 locus. In contrast, if the transgenic construct does not contain additional selectable traits and relies on negative selection at the SNR1 locus, the efficiency of successful transgenesis largely depends on the efficiency of cotransfection of the CRISPR plasmid and the transgenic construct.
Second, although CRISPR/Cas9 mediated site-specific integration of a non-homologous DNA fragment is frequently used for complementation and transgenesis, it should be noted that the orientation of insertion cannot be guaranteed in such cases as either direction is possible. This may pose problems to some applications. For example, to complement a mutant with different gene alleles, it is difficult to ensure that all alleles are inserted in the same orientation, and discrepancies in orientation may cause expression differences. For this type of application, DNA constructs with sequences homologous to the SNR1 locus are recommended, which will drive the proper integration orientation (Figure 6).
Third, during transfection, the ratio between the CRISPR plasmid and the transgenic DNA molecule is critical for successful transgenic strain construction. This ratio needs to be adjusted according to the selection strategies. The following guidelines are recommended: 1) If the transgenic construct contains a drug resistant marker and the corresponding drug is the only selection used to generate transgenic parasites, i.e. sinefungin is not used, the suggested molar ratio between the transgenic construct and the CRISPR plasmid is 1:5. Using more CRISPR plasmid in this case increases the likelihood that parasites receiving the transgenic construct will also receive the CRISPR plasmid, therefore the drug resistant parasites are more likely to have the marker inserted at the CRISPR targeting site. If the ratio is reversed, most parasites that receive the transgenic construct won&#39;t get the CRISPR plasmid. As a consequence, the vast majority of drug resistant parasites obtain the transgenic construct through random integration not associated with CRISPR/CAS9 mediated site-specific insertion. 2) If the transgenic construct contains a drug resistant marker and the corresponding drug is used along with sinefungin to select transgenic parasites, the suggested molar ratio between the transgenic construct and the CRISPR plasmid is 1:1. This strategy provides the highest efficiency of transgenic strain construction. 3) If the transgenic construct does not contain a selectable maker, relying on negative selection by sinefungin alone to obtain transgenic parasites, the suggested molar ratio between the transgenic construct and the CRISPR plasmid is 5:1. The rationale for this design is the same as in the first guideline above. Since both pyrimethamine and sinefungin were used for selection in Protocol 5, the ratio between DHFR* mini gene and the SNR1 targeting CRISPR plasmid was set as 1:1.
Taken together, the methods outlined here have a level of detail that was not possible to convey in the original publication that identified SNR1 2. These protocols; specifically, the command line syntax, sequential layout of the programs utilized, and the use of CRISPR/Cas9 should aid future endeavors to identify new genes responsible for phenotypes.

Host range determines the extent of a parasites&#39; prevalence. Some parasites have very specific host requirements that limit the area from which they are found, others are generalists. One such generalist is Toxoplasma gondii (T. gondii). This parasite is found worldwide as it can infect all mammals and many birds. Humans are also susceptible and it is estimated that approximately 1/3 of the global population has been infected. Fortunately, a robust immune response normally controls the growth of the parasite, but in situations where the immune system is compromised the parasite can grow unchecked and cause diseases, often encephalic. Also, this parasite can cause congenital diseases if previously uninfected women are infected during pregnancy as they lack immune memory to quickly limit the spread of the parasite. Additionally, there is a burden of ocular toxoplasmosis that can result in vision loss 1. For these reasons T. gondii has become a focus of study, and due to the many molecular methods developed for its study, a model for Apicomplexan parasites. Two methods that will be discussed here are Quantitative Trait Locus (QTL) mapping and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing. QTL mapping and CRISPR/Cas9 editing are, respectively, forward and reverse genetic approaches that have been used in previous studies to identify and/or characterize T. gondii virulence genes. Here, these methods are combined to identify and confirm the function of a sinefungin resistance (SNFr) gene, TgME49_290860, and its orthologs (annotated as SNR1) 2.
Although T. gondii can infect a wide variety of intermediate hosts, it must pass through and infect the intestinal epithelial cells of a felid to complete the life cycle. Cats are the definitive hosts of the parasite where the sexual stages are produced and genetic recombination via meiosis occurs. To conduct a QTL study one must create a genetic cross, and in the case of Toxoplasma this means passing two different parasite strains that differ in a phenotypic trait, the parental stains, through a cat to produce recombinant progeny 3. Before being fed to cats, the parental strains are made resistant to separate drugs to allow for more efficient identification of recombinants by double drug selection of the progeny 4. Three drugs have been used in T. gondii for this purpose; fluorodeoxyribose (FUDR) for which uracil phosphoribosyl transferase (UPRT) is the resistance gene 5, adenosine arabinoside (ARA) for which Adenosine Kinase (AK) is the resistance gene 6, and sinefungin (SNF) for which the resistance gene was unknown 7. Several genetic crosses have been created for T. gondii, but only the 24 progeny of the ME49-FUDRr X VAND-SNFr cross were genotyped using whole genome sequencing (WGS) 8. This opened the possibility of mapping and identifying the SNF resistance gene using this cross as the VAND parent was made sinefungin resistant by chemical mutagenesis of the drug sensitive VAND (VAND-SNFs) strain, and the VAND reference genome was sequenced using the VAND-SNFs strain thus allowing for the identification of all polymorphisms between the progeny WGS and the VAND-SNFs reference genome, including the inherited parental VAND-SNFr mutation that rendered some of the progeny sinefungin resistant.
In order to identify the causal single nucleotide polymorphism (SNP) in the SNFr progeny, several computational based open source resources can be used to analyze the data. To create the genetic map for the ME49-FUDRr X VAND-SNFr cross the REDHORSE software suite 9 was developed that uses WGS alignments of the parents and progeny to accurately detect the genomic positions of genetic crossovers. This mapping information can then be combined with the phenotypic data (SNFr in the progeny) to format a dataset compatible with the &#39;qtl&#39; package 10 in the R statistical programming software where a QTL scan can be run to reveal significant loci correlated with the phenotype. To identify the causal SNP located within the QTL locus, the WGS reads of the progeny can be individually aligned to the sinefungin sensitive VAND genome using the Bowtie 2 alignment program 11 from which SNPs can be called using the VarScan mpileup2snp variant caller program 12. Using these SNPs, the QTL locus can then be scanned for polymorphisms that are present in the SNFr but not in the SNFs progeny. With the causal SNP identified in the coding region of a gene, genetic modification of the candidate SNFr gene can be performed in a SNFs strain to verify the drug resistance function.
The CRISPR/Cas9 genome editing system was recently established in Toxoplasma13, which added important tools for the exploration of the complex biology of this parasite, particularly for genetic studies in non-laboratory adapted strains. Because of the highly active Non-Homologous End Joining (NHEJ) activity in WT&#160;Toxoplasma cells, targeted genome modification is difficult to achieve since exogenously introduced DNA is randomly integrated to the genome at extremely high frequency 14. To increase the success rate of locus specific modification, different approaches have been employed to increase the efficiency of homologous recombination and/or decrease the NHEJ activity 15,16. One of these approaches is the CRISPR/Cas9 system. Compared to other methods, the CRISPR/Cas9 system is efficient at introducing site-specific modifications and is easy to design 13,17,18. In addition, it can be used in any Toxoplasma strain without extra modification to the parasite 13,19.
The CRISPR/Cas9 system originated from the adaptive immune system of Streptococcus pyogenes, which uses it to defend the invasion of mobile genetic elements such as phages 20,21,22. This system utilizes the RNA-guided DNA endonuclease enzyme Cas9 to introduce double-strand DNA break (DSB) into the target, which is subsequently repaired either by the error-prone NHEJ to inactivate target genes through short indel mutations, or by homology directed recombination to alter the target locus exactly as designed 23,24. The target specificity is determined by a small RNA molecule named single guide RNA (gRNA), which contains an individually designed 20 nt sequence that has 100% homology to the target DNA 22. The gRNA molecule also contains signatures recognized by Cas9 that guide the nuclease to the target site, which includes a special Protospacer Adjacent Motif (PAM, sequence is &#39;NGG&#39;) 25,26. Therefore, the gRNA molecule and the PAM sequence work together to determine the Cas9 cleavage site in the genome. One can easily change the gRNA sequence to target different sites for cleavage.
When the CRISPR/Cas9 system was first developed in Toxoplasma, a single plasmid expressing the Cas9 nuclease and the gRNA molecule was used to introduce DSB at the targeting site 13,17. It has been shown that the CRISPR/Cas9 system drastically increases the efficiency of site-specific genome modification, not only by homologous recombination, but also non-homologous integration of exogenous DNA 13. It does this in wildtype strains that contain NHEJ activity. Therefore, this system can be used in essentially any Toxoplasma strain for efficient genome editing. In a typical experiment, the target specific CRISPR plasmid and the DNA fragment used to modify the target are co-transfected into parasites. If the DNA fragment used to modify the target contains homologous sequences to the target locus, homologous recombination can be used to repair the DSB introduced by CRISPR/Cas9 to allow precise modification of the target. On the other hand, if the introduced DNA fragment does not contain homologous sequence, it can still be integrated into the CRISPR/Cas9 targeting site. The latter is often used to disrupt genes by insertion of selectable markers or to complement mutants at loci that allow negative selection 13. Here the SNR1 locus serves as an example to show how CRISPR/Cas9 can be used for gene disruption and the generation of transgenic parasites.

<table><tbody><tr><td>T25 flasks</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Human Foreskin Fibroblasts (HFF)</td><td>ATCC</td><td>SCRC-1041</td><td></td></tr><tr><td>&lt;em&gt;T. gondii&lt;/em&gt; ME49 strain</td><td>ATCC</td><td>50840</td><td></td></tr><tr><td>&lt;em&gt;T. gondii&lt;/em&gt;VAND strain</td><td>ATCC</td><td>PRA-344</td><td></td></tr><tr><td>DMEM (No Sodium Bicarbonate)</td><td>Life Sciences</td><td>12800017</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) Premium Grade</td><td>VWR International</td><td>97068-085</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp; 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>Gentamicin (10 mg/mL)</td><td>Life Technologies&amp;nbsp;</td><td>15710064</td><td></td></tr><tr><td>Cell Scraper for Flasks</td><td>VWR International</td><td>10062-904</td><td></td></tr><tr><td>Syringe 10 mL</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Blunt needles 22 G x 1&quot;</td><td>BRICO Products</td><td>BN2210</td><td></td></tr><tr><td>Nuclepore Filter 3.0 &amp;micro;m, 25 mm</td><td>GE Healthcare</td><td>110612</td><td></td></tr><tr><td>Swin-Lok Filter Holder 25 mm</td><td>GE Healthcare</td><td>420200</td><td></td></tr><tr><td>Hemacytometer</td><td>Propper MFG</td><td>90001</td><td></td></tr><tr><td>Sinefungin</td><td>Enzo Life Sciences</td><td>380-070-M001</td><td></td></tr><tr><td>R&amp;nbsp;</td><td>The R Foundation</td><td>https://www.r-project.org/</td><td></td></tr><tr><td>J/qtl</td><td>The Churchill Group</td><td>http://churchill.jax.org/software/jqtl.shtml</td><td></td></tr><tr><td>Bowtie2</td><td>John Hopkins University</td><td>http://bowtie-bio.sourceforge.net/bowtie2/index.shtml</td><td></td></tr><tr><td>NCBI SRA Toolkit</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/Traces/sra/?view=toolkit_doc</td><td></td></tr><tr><td>SAMtools</td><td>Wellcome Trust Sanger Institute</td><td>http://www.htslib.org/</td><td></td></tr><tr><td>VarScan</td><td>Washington University, St Louis</td><td>http://varscan.sourceforge.net/</td><td></td></tr><tr><td>MUMmer</td><td>JCVI &amp;amp; Univ Hamburg</td><td>http://mummer.sourceforge.net/</td><td></td></tr><tr><td>pSAG1::CAS9-U6::sgUPRT</td><td>Addgene</td><td>Plasmid #54467</td><td>&lt;em&gt;T. gondii&lt;/em&gt; CRISPR plasmid to cut the UPRT gene</td></tr><tr><td>pSAG1::CAS9-U6::sg290860-6</td><td>Addgene</td><td>Plasmid #59855</td><td>&lt;em&gt;T. gondii&lt;/em&gt; CRISPR plasmid to cut the TG*_290860 SNR1 gene</td></tr><tr><td>pUPRT::DHFR-D</td><td>Addgene</td><td>Plasmid #58528</td><td>Template for &lt;em&gt;DHFR*&lt;/em&gt; mini gene</td></tr><tr><td>Q5 Site-Directed Mutagenesis Kit&amp;nbsp;</td><td>New England Biolabs</td><td>E0552S</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>DF0446-07-5</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>746495</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P5504</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M1028</td><td></td></tr><tr><td>Adenosine triposhpate (ATP)</td><td>Sigma Aldrich</td><td>A6419</td><td></td></tr><tr><td>L-glutathione (GSH)</td><td>Sigma Aldrich</td><td>G4251</td><td></td></tr><tr><td>ECM 830 Electroporation System</td><td>BTX</td><td>45-0002</td><td></td></tr><tr><td>Electroporation Cuvette&amp;nbsp; 4 mm</td><td>Harvard Apparatus</td><td>45-0126</td><td></td></tr><tr><td>Crytal Violet</td><td>Alfa Aesar</td><td>B21932-14</td><td></td></tr><tr><td>6-well TC plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>24-well TC plate</td><td>Corning</td><td>353935</td><td></td></tr><tr><td>Cover glass 12 mm round</td><td>VWR International</td><td>89015-724</td><td></td></tr><tr><td>96-well TC plate</td><td>Corning</td><td>353075</td><td></td></tr><tr><td>Gibson Assembly Cloning Kit (Multi-fragment)</td><td>New England Biolabs</td><td>E5510S</td><td></td></tr><tr><td>Q5 High-Fidelity Polymerase&amp;nbsp;</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>Pyrimethamine</td><td>TCI AMERICA</td><td>P2037-1G</td><td>Use cuation when using pyrimethamine resistant parasites - see Protocol</td></tr></tbody></table>

qtl mapping and crispr/cas9 editing to identify a drug resistance gene in toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

This article outlines in detail several methods that can be used in succession to identify a gene responsible for drug resistance (Figure 1). The 24 progeny&#160;of the ME49-FUDRr X VAND-SNFr cross were assessed for resistance to the drug sinefungin as described in Protocol 1. Using the genetic map and the SNFr phenotype of the progeny, a QTL scan was run in R/qtl - Protocol 2 (Figure 2). This resulted in one significant peak on chromosome IX spanning approximately 1 Mbp. It is in this region that the causal mutation is located.
To identify the causal mutation, WGS reads from the progeny were aligned to the VAND-SNFs reference genome using Bowtie2 - Protocol 3.1, SNPs were called using VarScan mpileup2snp - Protocol 3.2, and the QTL locus in the VAND genome was identified using MUMmer - Protocol 3.3. Progeny SNPs within the QTL locus were extracted and scanned for a pattern where the SNFr progeny have a SNP and the SNFs do not, which is feasible because progeny SNPs were obtained by comparison to the VAND-SNFs reference genome (Figure 3). Only one SNP matched this pattern that results in an early stop codon in a putative amino acid transporter gene named SNR1.
Confirmation that SNR1 is the SNFr resistance gene was performed using the CRISPR/Cas9 system. A new CRISPR/Cas9 plasmid engineered for T. gondii gene editing was made containing a gRNA targeting the SNR1 gene near the SNFr mutation identified in the progeny - Protocol 4 (Figure 4A). The SNR1 targeting CRISPR/Cas9 plasmid was electroporated into a SNFs WT parasite strain and resistant mutants were obtained when cultured in 0.3 &#181;M sinefungin. No SNFr parasites were obtained when electroporated with the UPRT targeting CRISPR/Cas9 plasmid. Several SNFr CRISPR mutants were cloned and the region around the SNR1 gRNA target was sequenced. Each mutant had an indel that disrupted the coding sequence of the SNR1 gene (Figure 4B). This method can also be used to insert a targeting construct into the SNR1 locus via NHEJ (Figure 5), or by HR (Figure 6) - Supplemental File 2.
<img alt="Figure 1" src="/files/ftp_upload/55185/55185fig1.jpg" /> 
Figure 1: Schematic Workflow for Experiments Outlined in the Protocols. The main steps in identifying and confirming the SNFr gene using the ME49-FUDRr X VAND-SNFr cross are shown with reference to the appropriate Protocols outlined in this article. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55185/55185fig2.jpg" /> 
Figure 2: R/qtl Commands to run QTL Scan on the SNFr Phenotype. The commands as outlined in Protocol 2 were run in R using the qtl package. Representative commands and plot are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55185/55185fig3.jpg" /> 
Figure 3: Location of the Causal SNP. Progeny WGS reads were aligned to the VAND-SNFs reference genome with Bowtie 2, SNPs were called with VarScan, and the corresponding VAND QTL locus identified with MUMmer. SNPs located within the QTL locus were imported into a spreadsheet and the causal SNP was identified. SNFr progeny (yellow), SNFs progeny (green), SNFr SNP (red) (see Data File 3). <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55185/55185fig4.jpg" /> 
Figure 4: Confirming SNR1 as SNFr Gene using CRISPR/Cas9. (A) CRISPR/Cas9 induced indel mutations (green) in the SNR1 locus. (B) Representative indels in SNR1 caused by two different SNR1 specific CRISPR plasmids (C5 and C6 respectively). RH is the WT strain and C5 and C6 are SNFr mutants. (B is taken from reference 2). <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55185/55185fig5.jpg" /> 
Figure 5. Insertional Mutagenesis using CRISPR/Cas9. (A) Insertion of complementing or transgenic genes into the SNR1 locus by CRISPR/Cas9 mediated site-specific integration. Two possible orientations of the inserted DHFR* mini gene and the primers used for identification, F1/2 and R1/2 represent the priming sites of oligos used in diagnostic PCRs. (B) Diagnostic PCRs of one snr1::DHFR* clone, RH is used as a WT control. PCR of the GRA1p is included as a control checking the quality of genomic DNA as templates. Asterisks indicate lanes with unspecific bands (Figure 5B is taken from reference 2). <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55185/55185fig6.jpg" /> 
Figure 6: Gene Knockout using CRISPR/Cas9. A classic design for CRISPR/Cas9 mediated homologous gene replacement in T. gondii. The orientation of inserted transgene is fixed in this case. F1/R1,F2/R2 and F3/R3 represent the priming sites of oligos used in diagnostic PCRs. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/55185fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Data File 1: ME49-FUDRr X VAND-SNFr Cross File for use in R/qtl, &#34;csv&#34; Format.&#160; One .csv file that can be loaded into R/qtl with the format=&#34;csv&#34; option. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/Data_File_1.csv" target="_blank">Please click here to download this file.</a>
Data File 2: (chrid txt, genotype txt, markerpos.txt, mname.txt, phenonames.txt, and phenos.txt). ME49-FUDRr X VAND-SNFr Cross Files for use in R/qtl, &#34;gary&#34; Format. Six .txt files that can be loaded in R/qtl with the format=&#34;gary&#34; option. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/Data_File_2.zip" target="_blank">Please click here to download this file.</a>
Data File 3. Spreadsheet with Progeny SNPs across the SNFr QTL Locus. Contains one worksheet with the progeny SNPs, and a second worksheet with the phenotypic data of the parents and progeny of the cross. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/Data_File_3.xlsx" target="_blank">Please click here to download this file.</a>
Supplemental File 1: Additional Protocol Details. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/Supplementary_File_1_-_101916.docx" target="_blank">Please click here to download this file.</a>
Supplemental File 2: Protocol 5 - Utilization of Negative Selection at the SNR1&#160;Locus for Genetic Complementation or Transgenic Strain Construction. <a href="http://cloudflare.jove.com/files/ftp_upload/55185/Supplementary_File_2_-_101916.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii at JoVE.com

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

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16.2K Views.  Huazhong Agricultural University.  Details worden gepresenteerd over hoe QTL mapping met een genoomkaart op basis van een genoom sequentie kan worden gebruikt om een ​​drug resistentie gen in Toxoplasma gondii te identificeren en hoe dit kan worden gecontroleerd met het CRISPR / Cas9 systeem dat efficiënt een genomisch doel bewerkt, in dit geval de Drug resistentie gen. 

Video: QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

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

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

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

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

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 ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

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.

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.

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

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Immunology and Infection

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 ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Identifying Kinases Rewiring in Malaria Parasite: A Chemical Genetics Approach

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

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