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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

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  1. CRISPR-Cas9 Mediated Phage Therapy Provides a Sequence-Specific Alternative to Antibiotics CU Boulder

  2. Limitations of Current Antibacterial Treatments: the Post-Antibiotic Era

  3. Limitations of Current Antibacterial Treatments: Antibiotics Lack Specificity Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria

  4. Limitations of Current Antibacterial Treatments: Cannot Control Dose in Phage Therapy Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria

  5. CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage CRISPR- Cas9 Target bacteria

  6. CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 Target bacteria

  7. CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome PAM Target bacteria

  8. CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome DSB genome Target bacteria

  9. CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage gRNA CRISPR- Cas9 Cas9 genome DSB genome Target bacteria Cell death

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Can Targeted CRISPR-Cas9 Kill When Transformed Into Cells? targeting E. coli (kan+) Selected on Transform Chloramphenicol non- targeting E. coli (kan+)

  18. CRISPR-Cas9 Specifically Kills Target Cells Non-targeting gRNA Targeting gRNA 1920 colonies 8 colonies Grown on Chloramphenicol

  19. 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

  20. Phage Offers an Effective Delivery Mechanism Capsid Binding Proteins Packaging Signal Replication Packaging Protein expression Phage Genome Bacterial cell

  21. 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

  22. How We Manufactured a Replication- Deficient Phage Delivery System Replication Helper Phagemid Protein expression Packaging Replication Litmus28i Phagemid Bacterial cell

  23. Does Phage Preferentially Take Up Phagemid with an Intact Packaging Signal? Ampicillin Resistant Ampicillin Infection E. coli (F’) Kanamycin Resistant Kanamycin

  24. Phagemid is Preferentially Packaged Compared to Helper Phage Kanamycin Ampicillin Helper Phagemid Litmus28i phagemid 8 colonies 2056 colonies

  25. 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

  26. 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’)

  27. Packaging Signal is Necessary and Sufficient for Phagemid Packaging pSB1C3- packaging signal pSB1C3- amilCP Successful packaging No packaging

  28. 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

  29. 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

  30. 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’)

  31. CRISPR-Cas9 Mediated Phage Kills Bacteria Non-targeting gRNA Targeting gRNA 143 colonies 11 colonies Grown on Chloramphenicol

  32. 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

  33. Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance

  34. Incorrect Phagemid Packaging

  35. Insertion ensures pure phage product

  36. Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance

  37. Accounting for mutation by target diversification: Protospacer mutation block CRISPR-Cas9

  38. Accounting for mutation by target diversification: Multiple CRISPRs with unique spacers Target Genome

  39. Additional Considerations ● Increase proficiency of phage packaging ● Accounting for mutation in target organism ● Prevent proliferation of antibiotic resistance

  40. 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

  41. Outreach Wt + Resistant Strain mutant

  42. The End of the Antibiotic Era

  43. 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

  44. Supplementals

  45. Sequencing Phagemids from Surviving Colonies

  46. Target One Strain in a Mixed Population E.coli gRNA targets E.coli KanR KanamycinR lacZ CRISPR- Cas9 Infection X-gal and Chloramphenicol

  47. 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

  48. 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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