Difference between revisions of "Team:Tufts"
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The CRISPR-Cas9 system is a revolutionary new tool in molecular biology, allowing for double-stranded DNA breaks to be made almost anywhere in the genome. This technology makes it possible to alter targeted genes in an organism with the inclusion of a guide RNA that specifies where the Cas9 endonuclease cuts. CRISPR/Cas9 can be repurposed as a therapeutic gene-editing platform to treat genetic disorders, by replacing a mutated gene with a functional version. | The CRISPR-Cas9 system is a revolutionary new tool in molecular biology, allowing for double-stranded DNA breaks to be made almost anywhere in the genome. This technology makes it possible to alter targeted genes in an organism with the inclusion of a guide RNA that specifies where the Cas9 endonuclease cuts. CRISPR/Cas9 can be repurposed as a therapeutic gene-editing platform to treat genetic disorders, by replacing a mutated gene with a functional version. | ||
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The main technical hurdle remains the efficient delivery of the CRISPR-Cas9 platform into somatic cells. To mediate this delivery, we aim to leverage the cellular penetration capabilities of toxin B from the Clostridium difficile bacteria, which is capable of entering epithelial cells. The toxin will be rendered completely safe with several mutations, but will exist as a fusion protein with Cas9 bound to its guide RNA. This fusion protein should be able to enter somatic cells with the membrane binding domain of the C. difficile toxin B. Once crossing the cell membrane, the Cas9 domain of the fusion protein will ideally localize to the nucleus and be free to bind to the DNA of the cell at a locus specified by the gRNA complex. | The main technical hurdle remains the efficient delivery of the CRISPR-Cas9 platform into somatic cells. To mediate this delivery, we aim to leverage the cellular penetration capabilities of toxin B from the Clostridium difficile bacteria, which is capable of entering epithelial cells. The toxin will be rendered completely safe with several mutations, but will exist as a fusion protein with Cas9 bound to its guide RNA. This fusion protein should be able to enter somatic cells with the membrane binding domain of the C. difficile toxin B. Once crossing the cell membrane, the Cas9 domain of the fusion protein will ideally localize to the nucleus and be free to bind to the DNA of the cell at a locus specified by the gRNA complex. | ||
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Currently, we have received the plasmids for the atoxic TcdB and the Cas9 protein and have begun culturing them in E. coli. We plan to express the modified toxin in B. megaterium and then deliver the fusion protein to human epithelial cells expressing GFP. We have designed a proof of concept experiment to test for effective delivery of the Cas9 protein in which we will attempt to remove the GFP coding region in epithelial cells via this proposed CRISPR/Cas9 system. We will measure the fluorescence of the cells after TcdB/Cas9 fusion protein has been delivered, hoping to see a decrease in fluorescence as a result of the GFP coding region being removed by Cas9. | Currently, we have received the plasmids for the atoxic TcdB and the Cas9 protein and have begun culturing them in E. coli. We plan to express the modified toxin in B. megaterium and then deliver the fusion protein to human epithelial cells expressing GFP. We have designed a proof of concept experiment to test for effective delivery of the Cas9 protein in which we will attempt to remove the GFP coding region in epithelial cells via this proposed CRISPR/Cas9 system. We will measure the fluorescence of the cells after TcdB/Cas9 fusion protein has been delivered, hoping to see a decrease in fluorescence as a result of the GFP coding region being removed by Cas9. | ||
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Revision as of 02:28, 21 July 2015
The CRISPR-Cas9 system is a revolutionary new tool in molecular biology, allowing for double-stranded DNA breaks to be made almost anywhere in the genome. This technology makes it possible to alter targeted genes in an organism with the inclusion of a guide RNA that specifies where the Cas9 endonuclease cuts. CRISPR/Cas9 can be repurposed as a therapeutic gene-editing platform to treat genetic disorders, by replacing a mutated gene with a functional version.
The main technical hurdle remains the efficient delivery of the CRISPR-Cas9 platform into somatic cells. To mediate this delivery, we aim to leverage the cellular penetration capabilities of toxin B from the Clostridium difficile bacteria, which is capable of entering epithelial cells. The toxin will be rendered completely safe with several mutations, but will exist as a fusion protein with Cas9 bound to its guide RNA. This fusion protein should be able to enter somatic cells with the membrane binding domain of the C. difficile toxin B. Once crossing the cell membrane, the Cas9 domain of the fusion protein will ideally localize to the nucleus and be free to bind to the DNA of the cell at a locus specified by the gRNA complex.
Currently, we have received the plasmids for the atoxic TcdB and the Cas9 protein and have begun culturing them in E. coli. We plan to express the modified toxin in B. megaterium and then deliver the fusion protein to human epithelial cells expressing GFP. We have designed a proof of concept experiment to test for effective delivery of the Cas9 protein in which we will attempt to remove the GFP coding region in epithelial cells via this proposed CRISPR/Cas9 system. We will measure the fluorescence of the cells after TcdB/Cas9 fusion protein has been delivered, hoping to see a decrease in fluorescence as a result of the GFP coding region being removed by Cas9.
The main technical hurdle remains the efficient delivery of the CRISPR-Cas9 platform into somatic cells. To mediate this delivery, we aim to leverage the cellular penetration capabilities of toxin B from the Clostridium difficile bacteria, which is capable of entering epithelial cells. The toxin will be rendered completely safe with several mutations, but will exist as a fusion protein with Cas9 bound to its guide RNA. This fusion protein should be able to enter somatic cells with the membrane binding domain of the C. difficile toxin B. Once crossing the cell membrane, the Cas9 domain of the fusion protein will ideally localize to the nucleus and be free to bind to the DNA of the cell at a locus specified by the gRNA complex.
Currently, we have received the plasmids for the atoxic TcdB and the Cas9 protein and have begun culturing them in E. coli. We plan to express the modified toxin in B. megaterium and then deliver the fusion protein to human epithelial cells expressing GFP. We have designed a proof of concept experiment to test for effective delivery of the Cas9 protein in which we will attempt to remove the GFP coding region in epithelial cells via this proposed CRISPR/Cas9 system. We will measure the fluorescence of the cells after TcdB/Cas9 fusion protein has been delivered, hoping to see a decrease in fluorescence as a result of the GFP coding region being removed by Cas9.