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CU-Boulder Championship Presentation
CRISPR-Cas9 Mediated Phage Therapy Provides a Sequence-Specific Alternative to Antibiotics CU Boulder Limitations of Current Antibacterial Treatments: the Post-Antibiotic Era Limitations of Current Antibacterial Treatments: Antibiotics Lack Specificity Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria Limitations of Current Antibacterial Treatments: Cannot Control Dose in Phage Therapy Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage CRISPRCas9 Target bacteria CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA Cas9 CRISPRCas9 Target bacteria CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA Cas9 CRISPRCas9 genome PAM Target bacteria CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA Cas9 CRISPRCas9 genome DSB genome Target bacteria CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA Cas9 CRISPRCas9 genome DSB genome Cell death Target bacteria Identifying Species-Unique Target Sequences Kill ❏ ❏ ❏ ❏ ❏ Salmonella enterica Staphylococcus aureus Mycobacteriaceae tuberculosis Streptococcus pneumoniae Clostridium difficile Keep ❏ ❏ ❏ ❏ ❏ Escherichia coli Actinomyces viscosus Staphylococcus epidermidis Lactobacillus acidophilus Bacillus coagulans Identifying Species-Unique Target Sequences Kill ❏ ❏ ❏ ❏ ❏ Salmonella enterica Staphylococcus aureus Mycobacteriaceae tuberculosis Streptococcus pneumoniae Clostridium difficile CRISPR guide RNAs AGCCGGCCACAGUCGAUGAAUCCAGAAAAG CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG GGCGCCCCUGCGCUGACAGCCGGAACACGG AGUCAUAGCCGAAUAGCCUCUCCACCCAAG Keep ❏ ❏ ❏ ❏ ❏ Escherichia coli Actinomyces viscosus Staphylococcus epidermidis Lactobacillus acidophilus Bacillus coagulans Identifying Species-Unique Target Sequences Kill ❏ ❏ ❏ ❏ ❏ Salmonella enterica Staphylococcus aureus Mycobacteriaceae tuberculosis Streptococcus pneumoniae Clostridium difficile CRISPR guide RNAs AGCCGGCCACAGUCGAUGAAUCCAGAAAAG CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG GGCGCCCCUGCGCUGACAGCCGGAACACGG AGUCAUAGCCGAAUAGCCUCUCCACCCAAG Keep ❏ ❏ ❏ ❏ ❏ Escherichia coli Actinomyces viscosus Staphylococcus epidermidis Lactobacillus acidophilus Bacillus coagulans Not found in keeps? No Yes No No No Identifying Species-Unique Target Sequences Kill ❏ ❏ ❏ ❏ ❏ Keep Salmonella enterica Staphylococcus aureus Mycobacteriaceae tuberculosis Streptococcus pneumoniae Clostridium difficile ❏ ❏ ❏ ❏ ❏ Escherichia coli Actinomyces viscosus Staphylococcus epidermidis Lactobacillus acidophilus Bacillus coagulans Optimal gRNA CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG Project Aims • Demonstrate sequence specific CRISPRCas9 killing • Quantify efficiency of helper phagemid system • Determine if packaging signal functions on pSB1C3 construct • Show that CRISPR-Cas9 harboring phage are programmable, sequence-specific antimicrobials Design of a gRNA to Target kan Resistance Kill ❏ Escherichia coli K-12 (kan+) Keep ❏ Escherichia coli K-12 ❏ Escherichia coli MG1655 Optimal gRNA GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG Modification of Stanford-Brown Part to Target Kanamycin Resistance Gene cas9 nontargeting pSB1C3 Part BBa_K1218011 Stanford-Brown 2013 CRISPR change spacer targeting targeting CRISPR Can Targeted CRISPR-Cas9 Kill When Transformed Into Cells? targeting E. coli (kan+) Selected on Chloramphenicol Transform nontargeting E. coli (kan+) CRISPR-Cas9 Specifically Kills Target Cells Non-targeting gRNA 1920 colonies Targeting gRNA 8 colonies Grown on Chloramphenicol Project Aims • Demonstrate sequence specific CRISPRCas9 killing • Quantify efficiency of helper phagemid system • Determine if packaging signal functions on pSB1C3 construct • Show that CRISPR-Cas9 harboring phage are programmable, sequence-specific antimicrobials Phage Offers an Effective Delivery Mechanism Capsid Binding Proteins Packaging Signal Phage Genome Replication Packaging Protein expression Bacterial cell How We Manufactured a Replication Deficient Phage Structural genes LItmus28i phagemid Packaging signal Litmus28i Phagemid Bacterial cell Helper Phagemid Disrupted packaging signal Helper Phagemid Litmus28i Phagemid Bacterial cell How We Manufactured a ReplicationDeficient Phage Delivery System Helper Phagemid Litmus28i Phagemid Replication Protein expression Packaging Replication Bacterial cell Does Phage Preferentially Take Up Phagemid with an Intact Packaging Signal? Ampicillin Resistant Ampicillin Infection E. coli (F’) Kanamycin Resistant Kanamycin Phagemid is Preferentially Packaged Compared to Helper Phage Ampicillin Litmus28i phagemid 2056 colonies Kanamycin Helper Phagemid 8 colonies Project Aims • Demonstrate sequence specific CRISPRCas9 killing • Quantify efficiency of helper phagemid system • Determine if packaging signal functions on pSB1C3 construct • Show that CRISPR-Cas9 harboring phage are programmable, sequence-specific antimicrobials Is Packaging Signal Sufficient for Plasmid Delivery by Phage? packaging signal Part BBa_K1445000 CU-Boulder 2014 E. coli (F’) Packaging Selected on Chloramphenicol Infect amilCP E. coli (F’) Packaging Signal is Necessary and Sufficient for Phagemid Packaging pSB1C3- packaging signal Successful packaging pSB1C3- amilCP No packaging Project Aims • Demonstrate sequence specific CRISPRCas9 killing • Quantify efficiency of helper phagemid system • Determine if packaging signal functions on pSB1C3 construct • Show that CRISPR-Cas9 harboring phage are programmable, sequence-specific antimicrobials Modification of CRISPR-Cas9 BioBrick to Enable Packaging into Phage Cas9 packaging signal pSB1C3 nontargeting nontargeting CRISPR Part BBa_K1218011 Stanford-Brown 2013 targeting Part BBa_K1445001 CU-Boulder 2014 add packaging signal change gRNA targeting CRISPR Can Targeted CRISPR-Cas9 Kill When Delivered by Phage? targeting E. coli (kan+,F’) package into phage coats Infect Selected for infected cells on Chloramphenicol nontargeting E. coli (kan+,F’) CRISPR-Cas9 Mediated Phage Kills Bacteria Non-targeting gRNA 143 colonies Targeting gRNA 11 colonies Grown on Chloramphenicol Project Aims ü Demonstrate sequence specific CRISPRCas9 killing ü Quantify efficiency of helper phagemid system ü Determine if packaging signal functions on pSB1C3 construct ü Show that CRISPR-Cas9 harboring phage are programmable, sequence-specific antimicrobials Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance Incorrect Phagemid Packaging Insertion ensures pure phage product Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance Accounting for mutation by target diversification: Protospacer mutation block CRISPR-Cas9 Accounting for mutation by target diversification: Multiple CRISPRs with unique spacers Target Genome Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance Replace antibiotic resistance as selectable marker for phage production Insertion Excision trpC gene trpC gene Bacterial genome Ligation Bacterial genome (ΔtrpC) Transformation Phagemids (trpC+) trpC auxotroph Phage Outreach Wt+ Resistant Strain mutant The End of the Antibiotic Era Instructors Team Robin Dowell Anushree Chaterjee Josephina Hendrix Daren Kraft Leighla Tayefeh Kirill Novik Kendra Shattuck Joshua Ivie Rishabh Yadav Sarah Zimmermann Alexander Martinez Julissa Duran-Malle Justine Wagner Daniel Garey Andrea Mariani Advisors Tim Read Samantha O'Hara Michael Brasino Alexander Stemm-Wolf Cloe Pogoda Joe Rokicki Lavan Jhandan Supplementals Sequencing Phagemids from Surviving Colonies Target One Strain in a Mixed Population E.coli KanR gRNA targets KanamycinR E.coli lacZ CRISPRCas9 Infection X-gal and Chloramphenicol CRISPR-Cas9 Phage Has Benefits Over Antibiotics and Phage Therapy Considerations for an antibacterial Antibiotics Specific to target cell’s genome? X Fast development time? X Easy modification to new target? X Possible to control dose? Phage Therapy X Low cost of development? X No known side effects? X CRISPR-Cas9 Phage Can we Demonstrate CRISPR-Cas9 Mediated Killing of a Bacterial Cell? TGAGACCAGTCTCGGAAGCTCAAAGGTCTC GATAGAAGGCGATGCGCTGCGAATCGGGAG GATAGAAGGCGATGCGCTGCGAATCGGGAGCGG Scramble gRNA Targeting gRNA Target Sequence Cas9 endonuclease guide RNA Kanamycin Resistance gene GATAGAAGGCGATGCGCTGCGAATCGGGAGCGG target sequence PAM
