Limits...
CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi.

Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL - MBio (2014)

Bottom Line: We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations.These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome.In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi, bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.

No MeSH data available.


Related in: MedlinePlus

Cas9-mediated eGFP disruption in T. cruzi epimastigotes and trypomastigotes. (A) Design of constructs for stable expression of eGFP and nucleus-localized Cas9 in T. cruzi. (B) Flow cytometric analysis results for T. cruzi eGFP- and Cas9-expressing epimastigotes transfected with eGFP sgRNA. Disruption of eGFP was evident as early as 2 days posttransfection (dpt), and progressive loss of the GFP signal was observed over time. (C) ELISA analysis results for Cas9 expression in GFP-positive (eGFP-intact parasites [sequence confirmed]) and GFP-negative (Cas9-induced KO) parasites sorted following eGFP disruption. Expression levels of Cas9 were normalized to α-tubulin expression in corresponding samples. (D) eGFP- and Cas9-expressing T. cruzi trypomastigotes transfected with eGFP sgRNA or control RNA and then used to infect Vero cells and imaged 5 days later. Comparison of 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei and kinetoplasts with GFP fluorescence demonstrated a mixture of GFP-expressing and nonexpressing parasites in the eGFP-targeting sgRNA-transfected group and uniform GFP expression in parasites transfected with control RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4281920&req=5

fig1: Cas9-mediated eGFP disruption in T. cruzi epimastigotes and trypomastigotes. (A) Design of constructs for stable expression of eGFP and nucleus-localized Cas9 in T. cruzi. (B) Flow cytometric analysis results for T. cruzi eGFP- and Cas9-expressing epimastigotes transfected with eGFP sgRNA. Disruption of eGFP was evident as early as 2 days posttransfection (dpt), and progressive loss of the GFP signal was observed over time. (C) ELISA analysis results for Cas9 expression in GFP-positive (eGFP-intact parasites [sequence confirmed]) and GFP-negative (Cas9-induced KO) parasites sorted following eGFP disruption. Expression levels of Cas9 were normalized to α-tubulin expression in corresponding samples. (D) eGFP- and Cas9-expressing T. cruzi trypomastigotes transfected with eGFP sgRNA or control RNA and then used to infect Vero cells and imaged 5 days later. Comparison of 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei and kinetoplasts with GFP fluorescence demonstrated a mixture of GFP-expressing and nonexpressing parasites in the eGFP-targeting sgRNA-transfected group and uniform GFP expression in parasites transfected with control RNA.

Mentions: To determine the ability of single guide RNA (sgRNA) of Cas9 to disrupt genes in T. cruzi, we first stably expressed both enhanced green fluorescent protein (eGFP) and Cas9 in T. cruzi by using separate pTrex backbone plasmids (9) under G418 and blasticidin drug selection, respectively (Fig. 1A). Transfection of epimastigotes of T. cruzi with sgRNA, which was previously shown to mediate eGFP disruption in human cell lines (10), resulted in rapid and highly efficient reduction in GFP expression. Each of the three sgRNAs induced loss of GFP in ~50 to 60% of parasites as early as day 2 after transfection (Fig. 1B), No reduction in eGFP expression was observed in epimastigotes transfected with 80-bp human 18S rRNA as a control. GFP-targeted sgRNAs were also very efficient at disrupting gene expression when electroporated into trypomastigotes of T. cruzi, with Vero cells infected with recently transfected trypomastigotes showing a mixture of GFP-positive and GFP-negative parasites 5 days after transfection/infection (Fig. 1D).


CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi.

Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL - MBio (2014)

Cas9-mediated eGFP disruption in T. cruzi epimastigotes and trypomastigotes. (A) Design of constructs for stable expression of eGFP and nucleus-localized Cas9 in T. cruzi. (B) Flow cytometric analysis results for T. cruzi eGFP- and Cas9-expressing epimastigotes transfected with eGFP sgRNA. Disruption of eGFP was evident as early as 2 days posttransfection (dpt), and progressive loss of the GFP signal was observed over time. (C) ELISA analysis results for Cas9 expression in GFP-positive (eGFP-intact parasites [sequence confirmed]) and GFP-negative (Cas9-induced KO) parasites sorted following eGFP disruption. Expression levels of Cas9 were normalized to α-tubulin expression in corresponding samples. (D) eGFP- and Cas9-expressing T. cruzi trypomastigotes transfected with eGFP sgRNA or control RNA and then used to infect Vero cells and imaged 5 days later. Comparison of 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei and kinetoplasts with GFP fluorescence demonstrated a mixture of GFP-expressing and nonexpressing parasites in the eGFP-targeting sgRNA-transfected group and uniform GFP expression in parasites transfected with control RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281920&req=5

fig1: Cas9-mediated eGFP disruption in T. cruzi epimastigotes and trypomastigotes. (A) Design of constructs for stable expression of eGFP and nucleus-localized Cas9 in T. cruzi. (B) Flow cytometric analysis results for T. cruzi eGFP- and Cas9-expressing epimastigotes transfected with eGFP sgRNA. Disruption of eGFP was evident as early as 2 days posttransfection (dpt), and progressive loss of the GFP signal was observed over time. (C) ELISA analysis results for Cas9 expression in GFP-positive (eGFP-intact parasites [sequence confirmed]) and GFP-negative (Cas9-induced KO) parasites sorted following eGFP disruption. Expression levels of Cas9 were normalized to α-tubulin expression in corresponding samples. (D) eGFP- and Cas9-expressing T. cruzi trypomastigotes transfected with eGFP sgRNA or control RNA and then used to infect Vero cells and imaged 5 days later. Comparison of 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei and kinetoplasts with GFP fluorescence demonstrated a mixture of GFP-expressing and nonexpressing parasites in the eGFP-targeting sgRNA-transfected group and uniform GFP expression in parasites transfected with control RNA.
Mentions: To determine the ability of single guide RNA (sgRNA) of Cas9 to disrupt genes in T. cruzi, we first stably expressed both enhanced green fluorescent protein (eGFP) and Cas9 in T. cruzi by using separate pTrex backbone plasmids (9) under G418 and blasticidin drug selection, respectively (Fig. 1A). Transfection of epimastigotes of T. cruzi with sgRNA, which was previously shown to mediate eGFP disruption in human cell lines (10), resulted in rapid and highly efficient reduction in GFP expression. Each of the three sgRNAs induced loss of GFP in ~50 to 60% of parasites as early as day 2 after transfection (Fig. 1B), No reduction in eGFP expression was observed in epimastigotes transfected with 80-bp human 18S rRNA as a control. GFP-targeted sgRNAs were also very efficient at disrupting gene expression when electroporated into trypomastigotes of T. cruzi, with Vero cells infected with recently transfected trypomastigotes showing a mixture of GFP-positive and GFP-negative parasites 5 days after transfection/infection (Fig. 1D).

Bottom Line: We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations.These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome.In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi, bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.

No MeSH data available.


Related in: MedlinePlus