Trypanosoma cruzi CRISPR/Cas9 System

Trypanosoma cruzi CRISPR/Cas9 System plasmids allow for the amplification of a specific sgRNA sequence or express cas9 to generate CRISPR-ablated, red/green fluorescent parasites.

Highlights:

  • CAS9/pTREX-n: S. pyogenes Cas9 sequence with a twice-repeated sequence of the simian virus 40 nuclear localization signal and GFP in the pTREX-n backbone. Used for cloning a specific sgRNA by BamHI, to be co-expressed with Cas9 for genome editing in Trypanosoma cruzi
  • pUC_sgRNA: Contains the empty sgRNA backbone sequence (tracrRNA, 82 bp) and is used as DNA template to amplify a specific sgRNA using a forward primer with the protospacer sequence for gene targeting
  • td Tomato/pTREX-b: Possesses tdTomato synthetic gene and blasticidin S deaminase (Bsd) gene and can be used for cloning a specific sgRNA by BamHI, and then transfect Trypanosoma cruzi epimastigotes expressing Cas9 (green fluorescent and neomycin resistant) to generate CRISPR-ablated, red/green fluorescent parasites

From the laboratory of Roberto Docampo, MD, PhD, University of Georgia.

The Investigator's Annexe Part of The Investigator's Annexe program.

Catalog Number Product DataSheet Size AVAILABILITY Price Qty
EGA031
CAS9/pTREX-n
spotted on filter paper In stock
Regular Price:$68.00
On Sale:
EGA032
pUC_sgRNA
spotted on filter paper In stock
Regular Price:$68.00
On Sale:
EGA033
tdTomato/pTREX-b
spotted on filter paper In stock
Regular Price:$68.00
On Sale:
Specifications

Product Type: Plasmid
Name: CAS9/pTREX-n
Gene/insert name: Fusion gene CAS9-HA-2xNLS-GFP
Antibiotic Resistance: Ampicillin
Fusion Tag(s): Fusion gene Cas9-HA-2xNLS-GFP
Grow in E. coli at 37 C: DH5alpha; 37C
Selectable markers: Neomycin (select with G418)
Cloning Site 5': XbaI
Cloning Site 3': HindIII
Insert Size: 4975 bp
Vector Backbone and Size: pTREX-n, backbone size without insert: 6227bp
High or low copy: High
Storage: -20C
Shipped: Ambient temperature

Documentation

Trypanosoma cruzi CRISPR/Cas9 System Plasmids

Plasmid: CAS9/pTREX-n PUC_sgRNA tdTomato/pTREX-b
Gene/Insert Name: Fusion gene CAS9-HA-2xNLS-GFP TracrRNA sequence tdTomato
Insert Size (bp): 4975 82 1430
Species: Synthetic; Streptococcus pyogenes N/A Synthetic
Fusion Proteins/Tags: Fusion gene Cas9-HA-2xNLS-GFP N/A
Vector Backbone and Size: pTREX-n, backbone size without insert: 6227bp pUCAmp; 3150bp pTREX-b, backbone size without insert: ~5868bp
Cloning Site 5': XbaI 5' Sequencing Primer: M13_fwd20_primer and M13_pUC_fwd_primer XbaI
Cloning Site 3': HindIII 3' Sequencing Primer: M13_rev_primer and M13_pUC_rev_primer HindIII
Antibiotic Resistance: Ampicillin Ampicillin Ampicillin
High or Low Copy: High High High
Grow in Standard E. coli @ 37C: DH5alpha; 37C DH5alpha; 37C DH5alpha; 37C
Selectable Markers: Neomycin (select with G418) Blasticidin
Storage Temperature: -20C -20C -20C
Shipped: Room Temperature Room Temperature Room Temperature
Plasmid Maps

CAS9/pTREX-n

Plasmid map for CAS9-HA-2xNLS-GFP in pTREX-n. Restriction map of molecular constructs generated for the CRISPR/Cas9 system in T. cruzi. Cas9/pTREX-n derived from vectors pTREX-n by insertion of the Cas9-HA-2 NLS-GFP fusion gene through XbaI and HindIII restriction sites.

tdTomato/pTREX-b

Plasmid map for td Tomato in pTREX-b_NL. Plasmid contains cytoplasmic red fluorescence and blasticidin resistance and can be used to transfect T. cruzi epimastigotes expressing Cas9 (green fluorescent and neomycin resistant) to generated CRISPR-ablated, red/green fluorescent parasites.

Adapted from: Lander N. et al., mBIO. 2015. 6(4): e01012-15.

Provider
From the laboratory of Roberto Docampo, MD, PhD, University of Georgia.
References
  1. Lander N, Li ZH, Niyogi S, Docampo R. CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment. mBIO. 2015. 6(4): e01012-15.

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