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CRISPR Plasmids: Parasites


Eukaryotic parasites continue to cause substantial death and disease in the world. Parasites such as Plasmodium (malaria), Toxoplasma (toxoplasmosis), Trypanosoma (African sleeping sickness), Cryptosporidium (cryptosporidiosis), Leishmania (leishmaniasis), and others have been the focus of biomedical research for decades. Recent developments with CRISPR-Cas9 gene editing have propelled both reverse and forward genetics of these parasites. Moreover, CRISPR-dCas9 gene regulation has also been established for some of these parasites. Together, these tools have enabled precise genetic analyses to pinpoint genes linked to parasite virulence, transmission, and development. This collection brings together some of the plasmids that have been used in these efforts.

The content for this page was generated with the help of Scott Lindner.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Plasmid Gene/Insert Selectable Marker PI Publication Hidden Extra Search Info
pU6-UniversalCas9 (Synthetic), Toxoplasma U6 upstream region - protospacer cloning sequence - chiRNA (Synthetic) Lourido Lourido lab Toxoplasma CRISPR Plasmid (unpublished) This plasmid can be used to mutate genes in Toxoplasma gondii using the CRISPR/Cas9 system after having an appropriate protospacer cloned into it. pX330
pTrex-b-NLS-hSpCas9NLS-hSpCas9Blasticidin Tarleton CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi. MBio. 2014 Dec 30;6(1). pii: e02097-14. doi: 10.1128/mBio.02097-14. Expression of Cas9 in T. cruzi pTrex-b
pLPhygCAS9Humanized Streptococcus pyogenes Cas9 from PX330 (Addgene) (Other)Hygromycin Matlashewski CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani. MBio. 2015 Jul 21;6(4). pii: e00861-15. doi: 10.1128/mBio.00861-15. Expresses CAS9 in Leishmania pLPHyg2
Cas9/pTREX-nFusion gene Cas9-HA-2xNLS-GFP from vector pMJ920 (Addgene) . (Synthetic)Neomycin (select with G418) Docampo CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment. MBio. 2015 Jul 21;6(4). pii: e01012-15. doi: 10.1128/mBio.01012-15. Expresses the fusion gene CAS9-HA-2xNLS-GFP in the pTREX-n backbone. This vector is used for cloning a specific sgRNA by BamHI, to be co-expressed with Cas9 for genome editing in Trypanosoma cruzi. pTREX-n
pCas9-CATCas9 (Other), Chloramphenicol acetyltransferaseCAT Lourido A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. Encodes Cas9 and chloramphenicol acetyltransferase (CAT) pU6-Universal
pU6-DecoyDecoy sgRNA Lourido A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. Encodes Cas9 and a CRISPR sgRNA that alleviates toxicity to Cas9 in Toxoplasma gondii pU6-Universal
pLdCNgRNA and Cas9 (Other)Neomycin (select with G418) Matlashewski Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms. mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb. Express gRNA and Cas9 in Leishmania with Neomycin resistance pSP72
pLdCHgRNA and Cas9 (Other)Hygromycin Matlashewski Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms. mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb. Express gRNA and Cas9 in Leishmania with Hygromycin resistance pSP72
pRPaCas9Cas9 (Other) Horn Inducible high-efficiency CRISPR-Cas9-targeted gene editing and precision base editing in African trypanosomes. Sci Rep. 2018 May 21;8(1):7960. doi: 10.1038/s41598-018-26303-w. Expresses Cas9 in T. brucei (2T1) cells pUC
pACT1:Cas9-GFP, U6:sgTKCas9-GFP (Other), U6-sgTK (Other) Sibley A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability. Cell Host Microbe. 2019 Jun 18. pii: S1931-3128(19)30252-5. doi: 10.1016/j.chom.2019.05.007. Expresses Cas9 fused with GFP and a sgRNA targeting the Cryptosporidium parvum TK gene pUC19
pACT1:Cas9-GFP, U6:sgUPRTCas9-GFP (Other), U6-sgUPRT (Other) Sibley A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability. Cell Host Microbe. 2019 Jun 18. pii: S1931-3128(19)30252-5. doi: 10.1016/j.chom.2019.05.007. Expresses Cas9 fused with GFP and a sgRNA targeting the Cryptosporidium parvum UPRT gene pUC19
pBM019sgRNA#1 (Synthetic), SpCas9 (Synthetic), Chloramphenicol acetyltransferase (Other)Chloramphenicol Lourido Optimizing Systems for Cas9 Expression in Toxoplasma gondii. mSphere. 2019 Jun 26;4(3). pii: 4/3/e00386-19. doi: 10.1128/mSphere.00386-19. Construct for generating stable Cas9-expressing Toxoplasma gondii pX330
pSL1433SpCas9 (Other)Pyrimethamine Lindner Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii. J Biol Chem. 2019 Jun 14;294(24):9555-9566. doi: 10.1074/jbc.RA118.007121. Epub 2019 May 1. pDEF-SpCas9::GFP, Empty Vector for RGR and HDR Template, PyBiP-minimal promoter, 3rd Generation pDEF

Libraries

Toxoplasma CRISPR Knockout Pooled Library - Toxoplasma gondii CRISPR genome-wide knockout gRNA pooled library designed with 10 guides against 8,158 predicted protein-coding genes.


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