CRISPR-Cas9 Mediated Phage Therapy Provides a Sequence-Specific - - PowerPoint PPT Presentation

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CRISPR-Cas9 Mediated Phage Therapy Provides a Sequence-Specific - - PowerPoint PPT Presentation

Jun 11, 2023 233 likes •717 views

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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SLIDE 1

CRISPR-Cas9 Mediated Phage Therapy Provides a Sequence-Specific Alternative to Antibiotics

CU Boulder

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SLIDE 2

Limitations of Current Antibacterial Treatments: the Post-Antibiotic Era

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SLIDE 3

Limitations of Current Antibacterial Treatments: Antibiotics Lack Specificity

Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria

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SLIDE 4

Limitations of Current Antibacterial Treatments: Cannot Control Dose in Phage Therapy

Commensal Bacteria Pathogenic Bacteria Antibiotic Resistant Bacteria

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SLIDE 5

CRISPR- Cas9

Target bacteria

CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage

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SLIDE 6

Cas9 gRNA

CRISPR- Cas9

Target bacteria

CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage

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SLIDE 7

Cas9 genome gRNA

CRISPR- Cas9

Target bacteria

CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage

PAM

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SLIDE 8

Cas9 genome DSB genome gRNA

CRISPR- Cas9

Target bacteria

CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage

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SLIDE 9

Cas9 genome DSB genome gRNA

Cell death

CRISPR- Cas9

Target bacteria

CRISPR-Cas9 Mediated Phage Therapy Kills through Genome Cleavage

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SLIDE 10

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

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SLIDE 11

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 CRISPR guide RNAs AGCCGGCCACAGUCGAUGAAUCCAGAAAAG CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG GGCGCCCCUGCGCUGACAGCCGGAACACGG AGUCAUAGCCGAAUAGCCUCUCCACCCAAG

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SLIDE 12

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 Not found in keeps? No Yes No No No CRISPR guide RNAs AGCCGGCCACAGUCGAUGAAUCCAGAAAAG CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG GGCGCCCCUGCGCUGACAGCCGGAACACGG AGUCAUAGCCGAAUAGCCUCUCCACCCAAG

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SLIDE 13

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

Optimal gRNA CGUGCUCGCUCGAUGCGAUGUUUCGCUUGG

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SLIDE 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

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SLIDE 15

Kill ❏ Escherichia coli K-12 (kan+) Keep ❏ Escherichia coli K-12 ❏ Escherichia coli MG1655

Design of a gRNA to Target kan Resistance

Optimal gRNA GAUAGAAGGCGAUGCGCUGCGAAUCGGGAG

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Modification of Stanford-Brown Part to Target Kanamycin Resistance Gene

non- targeting targeting

change spacer

Part BBa_K1218011 Stanford-Brown 2013

cas9 CRISPR

pSB1C3

targeting CRISPR

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SLIDE 17

Can Targeted CRISPR-Cas9 Kill When Transformed Into Cells?

Transform Selected on Chloramphenicol

  • E. coli

(kan+)

  • E. coli

(kan+)

targeting non- targeting

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SLIDE 18

Targeting gRNA 8 colonies Non-targeting gRNA 1920 colonies

CRISPR-Cas9 Specifically Kills Target Cells

Grown on Chloramphenicol

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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

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SLIDE 20

Phage Offers an Effective Delivery Mechanism

Replication Protein expression Packaging Signal Phage Genome Capsid Packaging Binding Proteins

Bacterial cell

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How We Manufactured a Replication Deficient Phage

Helper Phagemid

Packaging signal Disrupted packaging signal Structural genes

Helper Phagemid Litmus28i Phagemid

LItmus28i phagemid

Bacterial cell Bacterial cell

Litmus28i Phagemid

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SLIDE 22

How We Manufactured a Replication- Deficient Phage Delivery System

Bacterial cell Protein expression

Litmus28i Phagemid

Replication Replication Packaging

Helper Phagemid

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SLIDE 23

Does Phage Preferentially Take Up Phagemid with an Intact Packaging Signal?

  • E. coli

(F’)

Infection

Ampicillin Kanamycin Ampicillin Resistant Kanamycin Resistant

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SLIDE 24

Phagemid is Preferentially Packaged Compared to Helper Phage

Ampicillin Litmus28i phagemid 2056 colonies Kanamycin Helper Phagemid 8 colonies

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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

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SLIDE 26

Is Packaging Signal Sufficient for Plasmid Delivery by Phage?

packaging signal

amilCP

Packaging Infect Selected on Chloramphenicol

  • E. coli

(F’)

  • E. coli

(F’)

Part BBa_K1445000 CU-Boulder 2014

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SLIDE 27

Packaging Signal is Necessary and Sufficient for Phagemid Packaging

pSB1C3- packaging signal Successful packaging pSB1C3- amilCP No packaging

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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

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SLIDE 29

Modification of CRISPR-Cas9 BioBrick to Enable Packaging into Phage

non- targeting

change gRNA

Part BBa_K1218011 Stanford-Brown 2013

non- targeting

Part BBa_K1445001 CU-Boulder 2014

targeting

add packaging signal

Cas9 CRISPR

packaging signal

pSB1C3 targeting CRISPR

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SLIDE 30

Can Targeted CRISPR-Cas9 Kill When Delivered by Phage?

package into phage coats Infect Selected for infected cells on Chloramphenicol

  • E. coli

(kan+,F’)

  • E. coli

(kan+,F’)

targeting non- targeting

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SLIDE 31

CRISPR-Cas9 Mediated Phage Kills Bacteria

Non-targeting gRNA 143 colonies Targeting gRNA 11 colonies

Grown on Chloramphenicol

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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

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Additional Considerations

  • Increase proficiency of phage packaging
  • Accounting for mutation in target
  • rganism
  • Prevent proliferation of antibiotic

resistance

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Incorrect Phagemid Packaging

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Insertion ensures pure phage product

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Additional Considerations

  • Increase proficiency of phage packaging
  • Accounting for mutation in target
  • rganism
  • Prevent proliferation of antibiotic

resistance

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

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Target Genome

Accounting for mutation by target diversification: Multiple CRISPRs with unique spacers

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Additional Considerations

  • Increase proficiency of phage packaging
  • Accounting for mutation in target
  • rganism
  • Prevent proliferation of antibiotic

resistance

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SLIDE 40

Replace antibiotic resistance as selectable marker for phage production

Phage trpC gene Bacterial genome trpC gene Excision Insertion Ligation

Transformation

Bacterial genome (ΔtrpC) Phagemids (trpC+) trpC auxotroph

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SLIDE 41

Outreach

Resistant mutant Wt+ Strain

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The End of the Antibiotic Era

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Instructors

Robin Dowell Anushree Chaterjee

Advisors

Tim Read Samantha O'Hara Michael Brasino Alexander Stemm-Wolf Cloe Pogoda Joe Rokicki Lavan Jhandan

Team

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

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SLIDE 44

Supplementals

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Sequencing Phagemids from Surviving Colonies

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Target One Strain in a Mixed Population

E.coli lacZ E.coli KanR Infection gRNA targets KanamycinR

CRISPR- Cas9

X-gal and Chloramphenicol

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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

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Can we Demonstrate CRISPR-Cas9 Mediated Killing of a Bacterial Cell?

GATAGAAGGCGATGCGCTGCGAATCGGGAGCGG

GATAGAAGGCGATGCGCTGCGAATCGGGAGCGG Target Sequence GATAGAAGGCGATGCGCTGCGAATCGGGAG TGAGACCAGTCTCGGAAGCTCAAAGGTCTC Targeting gRNA

Cas9 endonuclease guide RNA Kanamycin Resistance gene

Scramble gRNA

target sequence PAM