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Team:CU-Boulder/Project/Design
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To test the concept of CRISPR-Cas9 mediated phage therapy, a three step experiment was needed. | To test the concept of CRISPR-Cas9 mediated phage therapy, a three step experiment was needed. | ||
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===CRISPR-Cas Vector Synthesis=== | ===CRISPR-Cas Vector Synthesis=== | ||
The first step was to design a spacer RNA sequence to specifically bind within a target gene but no where else in the ''E. coli'' genome. An RNA sequence was designed to be complementary to a unique sequence found within the neomycin phosphotransferase gene. Using restriction digestion and ligation the spacer sequence in BBa_K1218011(CRISPR-Cas9 plasmid) was replaced with the new targeting spacer sequence. | The first step was to design a spacer RNA sequence to specifically bind within a target gene but no where else in the ''E. coli'' genome. An RNA sequence was designed to be complementary to a unique sequence found within the neomycin phosphotransferase gene. Using restriction digestion and ligation the spacer sequence in BBa_K1218011(CRISPR-Cas9 plasmid) was replaced with the new targeting spacer sequence. | ||
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===Verification of CRISPR specificity=== | ===Verification of CRISPR specificity=== | ||
The second phase of the project was to transform the targeting CRISPR-Cas9 plasmid into BW23115(::kan) ''E. coli'' to verify that Cas9 can be directed to a specific sequence determined by the spacer and that double stranded breaks in the genome are sufficient to kill a bacterium. The CRISPR-Cas plasmid has been shown to effectively kill bacterial strains when expressed within targeted bacterial cells if the spacer is complementary to the target cell’s genome¹ ² ³. The specificity of CRISPR-Cas9 killing has been demonstrated to be specifically cytotoxic to a single nucleotide mutation, meaning strains of bacteria differing in a single SNP can be differentiated by the CRISPR guide RNA². However, creating an effective delivery mechanism for the CRISPR-Cas9 plasmid remains a large obstacle. | The second phase of the project was to transform the targeting CRISPR-Cas9 plasmid into BW23115(::kan) ''E. coli'' to verify that Cas9 can be directed to a specific sequence determined by the spacer and that double stranded breaks in the genome are sufficient to kill a bacterium. The CRISPR-Cas plasmid has been shown to effectively kill bacterial strains when expressed within targeted bacterial cells if the spacer is complementary to the target cell’s genome¹ ² ³. The specificity of CRISPR-Cas9 killing has been demonstrated to be specifically cytotoxic to a single nucleotide mutation, meaning strains of bacteria differing in a single SNP can be differentiated by the CRISPR guide RNA². However, creating an effective delivery mechanism for the CRISPR-Cas9 plasmid remains a large obstacle. | ||
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===Phage delivery of CRISPR-Cas9=== | ===Phage delivery of CRISPR-Cas9=== | ||
The third part of the project attempts to solve the delivery mechanism problem by using non-replicating recombinant bacteriophage. The non-replicating phage are created by infecting cells containing helper phagemids (plasmids coding for phage structural proteins), M13K07, with the CRISPR-Cas9 phagemid (a plasmid containing a phage packaging signal), BBa_K1445001. Similar projects have been published in Nature Biotechnology this year by Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York¹ and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology². The results of these studies are promising; however, further refinement of the phage delivery is required to increase virulency rates². | The third part of the project attempts to solve the delivery mechanism problem by using non-replicating recombinant bacteriophage. The non-replicating phage are created by infecting cells containing helper phagemids (plasmids coding for phage structural proteins), M13K07, with the CRISPR-Cas9 phagemid (a plasmid containing a phage packaging signal), BBa_K1445001. Similar projects have been published in Nature Biotechnology this year by Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York¹ and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology². The results of these studies are promising; however, further refinement of the phage delivery is required to increase virulency rates². | ||
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===References:=== | ===References:=== | ||
:1. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043 | :1. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043 | ||
:2. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011 | :2. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011 | ||
:3. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13. | :3. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13. | ||
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Latest revision as of 00:00, 18 October 2014
Contents |
Design
To test the concept of CRISPR-Cas9 mediated phage therapy, a three step experiment was needed.
CRISPR-Cas Vector Synthesis
The first step was to design a spacer RNA sequence to specifically bind within a target gene but no where else in the E. coli genome. An RNA sequence was designed to be complementary to a unique sequence found within the neomycin phosphotransferase gene. Using restriction digestion and ligation the spacer sequence in BBa_K1218011(CRISPR-Cas9 plasmid) was replaced with the new targeting spacer sequence.
Verification of CRISPR specificity
The second phase of the project was to transform the targeting CRISPR-Cas9 plasmid into BW23115(::kan) E. coli to verify that Cas9 can be directed to a specific sequence determined by the spacer and that double stranded breaks in the genome are sufficient to kill a bacterium. The CRISPR-Cas plasmid has been shown to effectively kill bacterial strains when expressed within targeted bacterial cells if the spacer is complementary to the target cell’s genome¹ ² ³. The specificity of CRISPR-Cas9 killing has been demonstrated to be specifically cytotoxic to a single nucleotide mutation, meaning strains of bacteria differing in a single SNP can be differentiated by the CRISPR guide RNA². However, creating an effective delivery mechanism for the CRISPR-Cas9 plasmid remains a large obstacle.
Phage delivery of CRISPR-Cas9
The third part of the project attempts to solve the delivery mechanism problem by using non-replicating recombinant bacteriophage. The non-replicating phage are created by infecting cells containing helper phagemids (plasmids coding for phage structural proteins), M13K07, with the CRISPR-Cas9 phagemid (a plasmid containing a phage packaging signal), BBa_K1445001. Similar projects have been published in Nature Biotechnology this year by Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York¹ and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology². The results of these studies are promising; however, further refinement of the phage delivery is required to increase virulency rates².
References:
- 1. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043
- 2. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011
- 3. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13.
