IP Library › Granted Patent US 12,246,061
Granted Patent B2
US 12,246,061 · App. 18/425,204 · Granted Mar 11, 2025

Sequence specific antimicrobials

Inventors: David Bikard (Paris, FR); Luciano Marraffini (Brooklyn, NY)
Assignee: The Rockefeller University
A61K38/465A01N63/00A61K31/7105A61K31/713A61K45/06C12N9/16C12N9/22C12N15/113C12N15/74C12Y301/00C12N2310/10C12N2310/20C12N2795/10331C12N2795/10332C12N2795/10343C12N2795/10371
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,246,061
App. No.
18/425,204
Granted
Mar 11, 2025
Kind
B2
Abstract

Provided are compositions and methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population, or for reducing any other type of unwanted bacteria in a mixed bacteria population. The compositions and methods involve targeting bacteria that are differentiated from other members of the population by at least one unique clustered regularly interspaced short palindromic repeats (CRISPR) targeted DNA sequence. The compositions and methods can be readily adapted to target any bacteria or any bacteria plasmid, or both.

Claims (32)

1. A method for selectively modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises at least a first and a second bacterial species, comprising:

(a) contacting the mixed population of bacteria with packaged, recombinant phagemids that are packaged in phage capsids, wherein the packaged phagemids comprise a clustered regularly interspaced short palindromic repeats (CRISPR) system comprising i) a type II CRISPR-associated enzyme and ii) two or more crRNA guides that target different chromosomal sequences in bacterial cells of the second bacterial species and,

(b) producing the crRNA guides in the bacterial cells of the second bacterial species, wherein the crRNA guides comprise i) a first crRNA guide complementary to a first target chromosomal sequence in the bacterial cells of the second bacterial species and ii) a second crRNA guide complementary to a second target chromosomal sequence in the bacterial cells of the second bacterial species, wherein the second target chromosomal sequence is different from the first target chromosomal sequence; and wherein:

1) the first target chromosomal sequence is comprised by a first antibiotic resistance gene and the second target chromosomal sequence is comprised by a second antibiotic resistance gene,

2) the first target chromosomal sequence is comprised by an antibiotic resistance gene and the second target chromosomal sequence is comprised by a virulence gene, or

3) the first target chromosomal sequence is comprised by a first virulence gene and the second target chromosomal sequence is comprised by a second virulence gene;

wherein the expressed type II CRISPR-associated enzyme cleaves the first and second target chromosomal sequences at the target sites of the crRNA guides in the bacterial cells of the second bacterial species,

wherein the first and second bacterial species are different and are selected from Streptococcus, Staphylococcus, Clostridium, Bacillus, Salmonella, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitidis , and Escherichia coli , and

wherein the growth of the bacterial cells of the first bacterial species in the mixed population is not inhibited, and wherein at least 50% of the bacterial cells of the second bacterial species are killed.

2. The method of claim 1 , wherein the mixed population of bacteria comprises E. coli.

3. The method of claim 1 , wherein the mixed population of bacteria comprises Streptococcus.

4. The method of claim 1 , wherein the mixed population of bacteria comprises Staphylococcus.

5. The method of claim 1 , wherein the mixed population of bacteria comprises Salmonella.

6. The method of claim 1 , wherein the mixed population of bacteria comprises Clostridium.

7. The method of claim 1 , wherein the second bacterial species is Staphylococcus.

8. The method of claim 7 , wherein the second bacterial species is Staphylococcus aureus.

9. The method of claim 8 , wherein the second bacterial species is a methicillin-resistant Staphylococcus aureus.

10. The method of claim 9 , wherein the first target chromosomal sequence is the mecA gene and the second target chromosomal sequence is the sek gene.

11. The method of claim 10 , wherein the type II Cas is a Cas9.

12. The method of claim 11 , wherein the type II Cas is a Streptococcus Cas9.

13. The method of claim 7 , wherein the first bacterial species is selected from Streptococcus, Clostridium, Bacillus, Salmonella, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitidis , and Escherichia coli.

14. The method of claim 13 , wherein the first bacterial species is Streptococcus.

15. The method of claim 13 , wherein the first bacterial species is Clostridium.

16. The method of claim 13 , wherein the first bacterial species is Bacillus.

17. The method of claim 13 , wherein the first bacterial species is Salmonella.

18. The method of claim 13 , wherein the first bacterial species is Helicobacter pylori.

19. The method of claim 13 , wherein the first bacterial species is Neisseria gonorrhoeae.

20. The method of claim 13 , wherein the first bacterial species is Neisseria meningitidis.

21. The method of claim 13 , wherein the first bacterial species is Escherichia coli.

22. The method of claim 1 , wherein the type II Cas is a Cas9.

23. The method of claim 22 , wherein the type II Cas is a Streptococcus Cas9.

24. The method of claim 23 , wherein at least 99.9% of the bacterial cells of the second bacterial species are killed.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 29, 2024
From: BIKARD, DAVID
To: THE ROCKEFELLER UNIVERSITY
Reel/Frame 066277/0788 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 29, 2024
From: MARRAFFINI, LUCIANO
To: THE ROCKEFELLER UNIVERSITY
Reel/Frame 066277/0848 →
Continuity (15)
Continuation 17168971 · Feb 5, 2021
Continuation 17088297 · Nov 3, 2020
Continuation 17088302 · Nov 3, 2020
Continuation 16877010 · May 18, 2020
Division 16877010 · May 18, 2020
Division 16877030 · May 18, 2020
Division 16877030 · May 18, 2020
Division 16877030 · May 18, 2020
Continuation 15159929 · May 20, 2016
Division 15159929 · May 20, 2016
Continuation 15159929 · May 20, 2016
Continuation 14766675
Division 14766675
Provisional Application 61761971 · Feb 7, 2013
Related Publication 20240165210A1 · May 23, 2024
References Cited (56)
US 8071352B2 · Yoon et al. · 2011 [cited by applicant]
US 9822372B2 · Zhang et al. · 2017 [cited by applicant]
US 10463049B2 · Clube et al. · 2019 [cited by applicant]
US 10506812B2 · Clube · 2019 [cited by applicant]
US 10524477B2 · Clube et al. · 2020 [cited by applicant]
US 10660943B2 · Bikard et al. · 2020 [cited by applicant]
US 11135273B2 · Bikard et al. · 2021 [cited by applicant]
US 11452765B2 · Bikard et al. · 2022 [cited by applicant]
US 11491209B2 · Bikard et al. · 2022 [cited by applicant]
US 11491210B2 · Bikard et al. · 2022 [cited by applicant]
US 11497797B2 · Bikard et al. · 2022 [cited by applicant]
US 11918631B2 · Bikard · 2024 [cited by examiner]
US 20030049841A1 · Short · 2003 [cited by applicant]
US 20090155768A1 · Scholl et al. · 2009 [cited by applicant]
US 20100076057A1 · Sontheimer et al. · 2010 [cited by applicant]
US 20110002889A1 · Barrangou et al. · 2011 [cited by applicant]
US 20140068797A1 · Doudna et al. · 2014 [cited by applicant]
US 20160115489A1 · Zhang et al. · 2016 [cited by applicant]
US 20170196225A1 · Clube et al. · 2017 [cited by applicant]
EP 2325332B1 · 2011 [cited by applicant]
WO 2012164565A1 · 2012 [cited by applicant]
WO 2013176772A1 · 2013 [cited by applicant]
Jiang, W., et al., RNA-Guided Editing of Bacterial Genomes Using CRISPR-Cas Systems, Nature Biotechnology, Jan. 29, 2013, vol. 31, pp. 233-241. [cited by applicant]
Westwater, C., et al., Use of Genetically Engineered Phage to Deliver Antimicrobial Agents to Bacteria: An Alternative Therapy for Treatment of Bacterial Infections, Antimicrobial Agents and Chemotherapy, Apr. 2003, vol… [cited by applicant]
Nakamura, S., et al., Metagenomic Diagnosis of Bacterial Infections, Emerging Infectious Diseases, Nov. 2008, vol. 14, pp. 1784-1786. [cited by applicant]
Bikard, D., et al., CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection, CellHost & Microbe, Aug. 15, 2012, vol. 12, 117-186. [cited by applicant]
Westwater, C., et al., Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria, Microbiology, 2002, vol. 148, pp. 943-950. [cited by applicant]
Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity, Science, Jun. 28, 2012, vol. 337, pp. 816-821. [cited by applicant]
Brouns, S.J.J., et al., Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes, Science, Aug. 15, 2008, vol. 321, pp. 960-964. [cited by applicant]
Cong, L., et al., Multiplex Genome Engineering Using CRISPR/Cas Systems, Science, Feb. 15, 2013, vol. 339, pp. 819-823. [cited by applicant]
Sapranauskas, R., et al., The [cited by applicant]
Edgar, R. & Qimron, U., The [cited by applicant]
Marraffini, L.A. & Sontheimer, E.J., CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA, Science, Dec. 19, 2008, vol. 332, pp. 1843-1845. [cited by applicant]
Garneau, J.E., et al., The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA, Nature, Nov. 4, Nov. 4, 2010, vol. 468, pp. 67-71. [cited by applicant]
Bae, T., et al., Prophages of [cited by applicant]
Third-Party Submission under 37 CFR 1.290 filed on Nov. 6, 2018 in U.S. Appl. No. 15/887,377, 5 pages. [cited by applicant]
Mei, J-M., et al., Identification of [cited by applicant]
Koonin, E.V., et al., Diversity, classification and evolution of CRISPR-Cas systems, Current Opinion in Microbiology, 2017, vol. 37, pp. 67-78. [cited by applicant]
Sorek, R., et al., CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea, Annual Review of Biochemistry, Mar. 11, 2013, pp. 237-266. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Jan. 27, 2020 for U.S. Appl. No. 14/766,675, 17 pages. [cited by applicant]
Fage, C., et al., Delivery of CRISPR-Cas systems using phage-based vectors, Current Opinion in Biotechnology, Dec. 23, 2020, vol. 68, pp. 174-180. [cited by applicant]
Makarova, K.S., et al., Evolution and classification of the CRISPR-Cas systems, Nature Reviews Microbiology, May 9, 2011, vol. 9, pp. 467-477. [cited by applicant]
United States Court of Appeals for the Federal Circuit, Judgment on Appeal from the United States Patent and Trademark Office, Patent Trial and Appeal Board in No. 106,132, Jul. 14, 2023, 1 page. [cited by applicant]
United States Court of Appeals for the Federal Circuit, Opinion on Appeal from the United States Patent and Trademark Office, Patent Trial and Appeal Board in No. 106,132, Jul. 14, 2023, 20 pages. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Mar. 2, 2017 for U.S. Appl. No. 14/766,675, 9 pages. [cited by applicant]
Final Rejection (Office Action) issued by United States Patent and Trademark Office on Oct. 11, 2018 for U.S. Appl. No. 14/766,675, 12 pages. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Apr. 18, 2019, for U.S. Appl. No. 14/766,675, 16 pages. [cited by applicant]
Hoyland-Kroghsbo, N.M., et al., A Quorum-Sensing-Induced Bacteriophage Defense Mechanism, mBio, Feb. 19, 2013, vol. 4, No. 1, e00362-12, pp. 1-8. [cited by applicant]
Del Pozo, J.L., et al., The Challenge of Treating Biofilm-associated Bacterial Infections, Translational Medicine, Aug. 2007, vol. 82, No. 2, pp. 204-209. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Jan. 24, 2020 for U.S. Appl. No. 15/159,929, 17 pages. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Sep. 18, 2017 for U.S. Appl. No. 15/159,929, 10 pages. [cited by applicant]
Final Rejection (Office Action) issued by United States Patent and Trademark Office on Apr. 27, 2018 for U.S. Appl. No. 15/159,929, 14 pages. [cited by applicant]
Declaration under 37 CFR 1.130(a) filed on Feb. 1, 2019 in U.S. Appl. No. 15/159,929, 2 pages. [cited by applicant]
Office Action Response and Request for Interference dated Feb. 18, 2020 in U.S. Appl. No. 15/159,929, 19 pages. [cited by applicant]
Redeclaration of Interference under 37 CFR 41.203(c) filed in United States Patent and Trademark Office in Interference No. 106,123 on Jul. 21, 2020, 6 pages. [cited by applicant]
Non-Final Rejection (Office Action) issued by United States Patent and Trademark Office on Apr. 2, 2019 for U.S. Appl. No. 15/159,929, 11 pages. [cited by applicant]