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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

Sequencing of the eGFP gene in GFP-disrupted clones sorted from parasite populations transfected with eGFP-targeting sgRNA 3 (top) and sgRNA 1 (bottom). Underlined is the sgRNA targeting sequence, the PAM is boxed (on the reverse strand), red marks show microhomology sequences flanking the sgRNA targeting sequence, and double-stranded cut sites are indicated with blue arrows. The sequence of negative clone 1 in the sgRNA-1 set (bottom) shows a single-base deletion in this region, likely due to a spontaneous mutation in GFP. All other sequences showed a 33-bp deletion; although deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site, as predicted by repair via an MMEJ pathway.
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fig3: Sequencing of the eGFP gene in GFP-disrupted clones sorted from parasite populations transfected with eGFP-targeting sgRNA 3 (top) and sgRNA 1 (bottom). Underlined is the sgRNA targeting sequence, the PAM is boxed (on the reverse strand), red marks show microhomology sequences flanking the sgRNA targeting sequence, and double-stranded cut sites are indicated with blue arrows. The sequence of negative clone 1 in the sgRNA-1 set (bottom) shows a single-base deletion in this region, likely due to a spontaneous mutation in GFP. All other sequences showed a 33-bp deletion; although deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site, as predicted by repair via an MMEJ pathway.

Mentions: Double-stranded DNA breaks induced by guided nucleases such as Cas9 are generally repaired in one of two ways: by error-prone NHEJ, resulting in indels of various sizes, or by homology-directed repair, which allows precise editing, from point mutations to large indels, depending on the available template. Sequencing of the eGFP gene from eGFP-negative clones produced by eGFP sequence-guided Cas9 DSBs showed a consistent 33-bp deletion at the sgRNA targeting site (Fig. 3). Although the deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site (Fig. 3, red highlight). This pattern is consistent with MMEJ pathway repair of DSBs in T. cruzi. Sequencing of the GFP gene from clones of the eGFP sgRNA-transfected population that remained GFP positive (Fig. 3) showed an intact GFP sequence, again consistent with insufficient Cas9-mediated DSBs in some parasites (data not shown).


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

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

Sequencing of the eGFP gene in GFP-disrupted clones sorted from parasite populations transfected with eGFP-targeting sgRNA 3 (top) and sgRNA 1 (bottom). Underlined is the sgRNA targeting sequence, the PAM is boxed (on the reverse strand), red marks show microhomology sequences flanking the sgRNA targeting sequence, and double-stranded cut sites are indicated with blue arrows. The sequence of negative clone 1 in the sgRNA-1 set (bottom) shows a single-base deletion in this region, likely due to a spontaneous mutation in GFP. All other sequences showed a 33-bp deletion; although deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site, as predicted by repair via an MMEJ pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Sequencing of the eGFP gene in GFP-disrupted clones sorted from parasite populations transfected with eGFP-targeting sgRNA 3 (top) and sgRNA 1 (bottom). Underlined is the sgRNA targeting sequence, the PAM is boxed (on the reverse strand), red marks show microhomology sequences flanking the sgRNA targeting sequence, and double-stranded cut sites are indicated with blue arrows. The sequence of negative clone 1 in the sgRNA-1 set (bottom) shows a single-base deletion in this region, likely due to a spontaneous mutation in GFP. All other sequences showed a 33-bp deletion; although deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site, as predicted by repair via an MMEJ pathway.
Mentions: Double-stranded DNA breaks induced by guided nucleases such as Cas9 are generally repaired in one of two ways: by error-prone NHEJ, resulting in indels of various sizes, or by homology-directed repair, which allows precise editing, from point mutations to large indels, depending on the available template. Sequencing of the eGFP gene from eGFP-negative clones produced by eGFP sequence-guided Cas9 DSBs showed a consistent 33-bp deletion at the sgRNA targeting site (Fig. 3). Although the deletion junctions occurred at slightly different positions, they all fell between a pair of homologous sequences of 10 bp flanking the cut site (Fig. 3, red highlight). This pattern is consistent with MMEJ pathway repair of DSBs in T. cruzi. Sequencing of the GFP gene from clones of the eGFP sgRNA-transfected population that remained GFP positive (Fig. 3) showed an intact GFP sequence, again consistent with insufficient Cas9-mediated DSBs in some parasites (data not shown).

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