Team:CU-Boulder/Project/Design

From 2014.igem.org

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==Design==
==Design==
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To test the concept of CRISPR-Cas9 mediated phage therapy, a three step experiment was needed.
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<br>
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===CRISPR-Cas Vector Synthesis===
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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===
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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===
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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:===
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: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
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:2.  Robert J Citorik, Mark Mimee, Timothy K Lu.  2014.  Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.  doi:10.1038/nbt.3011
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: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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{{Template:UCB-Footer}}

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.




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