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 CRISPR- Cas9 Target bacteria
CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 Target bacteria
CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome PAM Target bacteria
CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome DSB genome Target bacteria
CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome DSB genome Target bacteria Cell death
Identifying Species-Unique Target Sequences Kill Keep ❏ Salmonella enterica ❏ Escherichia coli ❏ Staphylococcus aureus ❏ Actinomyces viscosus ❏ Mycobacteriaceae tuberculosis ❏ Staphylococcus epidermidis ❏ Streptococcus pneumoniae ❏ Lactobacillus acidophilus ❏ Clostridium difficile ❏ Bacillus coagulans
Identifying Species-Unique Target Sequences Kill Keep ❏ Salmonella enterica ❏ Escherichia coli ❏ Staphylococcus aureus ❏ Actinomyces viscosus ❏ Mycobacteriaceae tuberculosis ❏ Staphylococcus epidermidis ❏ Streptococcus pneumoniae ❏ Lactobacillus acidophilus ❏ Clostridium difficile ❏ Bacillus coagulans CRISPR guide RNAs AGCCGGCCACAGUCGAUGAAUCCAGAAAAG CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG GGCGCCCCUGCGCUGACAGCCGGAACACGG AGUCAUAGCCGAAUAGCCUCUCCACCCAAG
Identifying Species-Unique Target Sequences Kill Keep ❏ Salmonella enterica ❏ Escherichia coli ❏ Staphylococcus aureus ❏ Actinomyces viscosus ❏ Mycobacteriaceae tuberculosis ❏ Staphylococcus epidermidis ❏ Streptococcus pneumoniae ❏ Lactobacillus acidophilus ❏ Clostridium difficile ❏ Bacillus coagulans CRISPR guide RNAs Not found in keeps? AGCCGGCCACAGUCGAUGAAUCCAGAAAAG No CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG Yes GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG No GGCGCCCCUGCGCUGACAGCCGGAACACGG No AGUCAUAGCCGAAUAGCCUCUCCACCCAAG No
Identifying Species-Unique Target Sequences Kill Keep ❏ Salmonella enterica ❏ Escherichia coli ❏ Staphylococcus aureus ❏ Actinomyces viscosus ❏ Mycobacteriaceae tuberculosis ❏ Staphylococcus epidermidis ❏ Streptococcus pneumoniae ❏ Lactobacillus acidophilus ❏ Clostridium difficile ❏ Bacillus coagulans Optimal gRNA CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG
Project Aims • Demonstrate sequence specific CRISPR- Cas9 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 Keep ❏ Escherichia coli K-12 ❏ Escherichia coli K-12 (kan + ) ❏ Escherichia coli MG1655 Optimal gRNA GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG
Modification of Stanford-Brown Part to Target Kanamycin Resistance Gene cas9 non- targeting targeting CRISPR CRISPR targeting change spacer pSB1C3 Part BBa_K1218011 Stanford-Brown 2013
Can Targeted CRISPR-Cas9 Kill When Transformed Into Cells? targeting E. coli (kan+) Selected on Transform Chloramphenicol non- targeting E. coli (kan+)
CRISPR-Cas9 Specifically Kills Target Cells Non-targeting gRNA Targeting gRNA 1920 colonies 8 colonies Grown on Chloramphenicol
Project Aims • Demonstrate sequence specific CRISPR- Cas9 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 Replication Packaging Protein expression Phage Genome Bacterial cell
How We Manufactured a Replication Deficient Phage Structural Helper genes Phagemid Disrupted packaging signal Helper Litmus28i LItmus28i Litmus28i Phagemid Phagemid Phagemid phagemid Packaging Bacterial cell Bacterial cell signal
How We Manufactured a Replication- Deficient Phage Delivery System Replication Helper Phagemid Protein expression Packaging Replication Litmus28i Phagemid 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 Kanamycin Ampicillin Helper Phagemid Litmus28i phagemid 8 colonies 2056 colonies
Project Aims • Demonstrate sequence specific CRISPR- Cas9 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 E. coli (F’) Part BBa_K1445000 Selected on Chloramphenicol CU-Boulder 2014 Packaging Infect amilCP E. coli (F’)
Packaging Signal is Necessary and Sufficient for Phagemid Packaging pSB1C3- packaging signal pSB1C3- amilCP Successful packaging No packaging
Project Aims • Demonstrate sequence specific CRISPR- Cas9 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 non- non- targeting CRISPR targeting CRISPR targeting targeting Part BBa_K1218011 Part BBa_K1445001 Stanford-Brown 2013 CU-Boulder 2014 add packaging change gRNA signal
Can Targeted CRISPR-Cas9 Kill When Delivered by Phage? targeting E. coli (kan+,F’) package into Infect Selected for infected cells on phage coats Chloramphenicol non- targeting E. coli (kan+,F’)
CRISPR-Cas9 Mediated Phage Kills Bacteria Non-targeting gRNA Targeting gRNA 143 colonies 11 colonies Grown on Chloramphenicol
Project Aims ü Demonstrate sequence specific CRISPR- Cas9 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 ( Δ trpC) Transformation Phagemids Phage (trpC + ) trpC auxotroph
Outreach Wt + Resistant Strain mutant
The End of the Antibiotic Era
I nstructors Team Robin Dowell Josephina Hendrix Anushree Chaterjee Daren Kraft Leighla Tayefeh Advisors Kirill Novik Kendra Shattuck Tim Read Joshua Ivie Samantha O'Hara Rishabh Yadav Michael Brasino Sarah Zimmermann Alexander Stemm-Wolf Alexander Martinez Cloe Pogoda Julissa Duran-Malle Joe Rokicki Justine Wagner Lavan Jhandan Daniel Garey Andrea Mariani
Supplementals
Sequencing Phagemids from Surviving Colonies
Target One Strain in a Mixed Population E.coli gRNA targets E.coli KanR KanamycinR lacZ CRISPR- Cas9 Infection X-gal and Chloramphenicol
CRISPR-Cas9 Phage Has Benefits Over Antibiotics and Phage Therapy Considerations for an antibacterial Antibiotics Phage Therapy CRISPR-Cas9 Phage Specific to target cell’s genome? X Fast development time? X Easy modification to new target? X Possible to control dose? X Low cost of development? X No known side effects? X
Can we Demonstrate CRISPR-Cas9 Mediated Killing of a Bacterial Cell? Scramble gRNA TGAGACCAGTCTCGGAAGCTCAAAGGTCTC Targeting gRNA GATAGAAGGCGATGCGCTGCGAATCGGGAG GATAGAAGGCGATGCGCTGCGAATCGGGAGCGG Target Sequence Cas9 endonuclease guide RNA Kanamycin GATAGAAGGCGATGCGCTGCGAATCGGGAG CGG Resistance gene PAM target sequence
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