Granted Patent B2

US 12,404,513 · App. 18/504,952 · Granted Sep 2, 2025

Inventors: Virginia Martinez (Copenhagen Ø, DK); Ruben Vazquez-Uribe (Copenhagen Ø, DK); Adam Takos (Copenhagen Ø, DK); Eric Van Der Helm (Copenhagen Ø, DK)

Assignee: SNIPR BIOME APS

C12N15/70A61K31/7088A61K38/465A61P3/04A61P31/04C07K14/33C12N9/22C12N15/11C12N2310/20C12N2800/80C12N2820/002C12N2820/007C12N2820/55C12N2830/005

Abstract

Claims (55)

1. A production strain bacterial cell comprising a nucleic acid vector for introduction into a target bacterial host cell for expression of Type I Cas3 and Cascade proteins in the target bacterial host cell, the vector comprising a first nucleotide sequence encoding a Type I Cas3 and a second nucleotide sequence encoding one or more cognate Cascade proteins, wherein the first nucleotide sequence is under the control of a promoter for controlling the expression of Type I Cas3 in the target bacterial host cell, wherein the promoter has a strength that is weaker than the Anderson Score strength of promoter BBa_J23108,

wherein the nucleic acid vector further comprises: (i) a CRISPR array for producing a crRNA in the target bacterial host cell; or (ii) a nucleotide sequence encoding a guide RNA (gRNA), wherein the crRNA or gRNA comprises a spacer sequence complementary to a target sequence of the target bacterial host cell, and

wherein the production strain bacterial cell does not comprise a crRNA or gRNA operable with the Cas3 to target and cut a chromosomal sequence of the production strain cell.

2. The production strain bacterial cell of claim 1 , wherein the target bacterial host cell is selected from the group consisting of Campylobacter, Burkholderia , and Acinetobacter.

3. The production strain bacterial cell of claim 2 , wherein the promoter is operable in a target host cell selected from the group consisting of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia stabilis, Burkholderia thailandensis, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter lwoffi , and Acinetobacter schindleri.

4. The production strain bacterial cell of claim 3 , wherein the Type I Cas3 and Cascade are E. coli, C. difficile, P. aeruginosa, K. pneumoniae, P. furiosus , or B. halodurans Cas and Cascade proteins.

5. The production strain bacterial cell of claim 1 , wherein the nucleic acid vector comprises an operon for expression of the Type I Cas3 and Cascade proteins, and:

(a) the first nucleotide sequence is between the promoter and the second nucleotide sequence in the operon;

(b) the operon comprises no Cas-encoding nucleotide sequences between the promoter and the first nucleotide sequence; or

(c) the operon comprises, in 5′ to 3′ direction, the promoter, the first nucleotide sequence, and the second nucleotide sequence.

6. The production strain bacterial cell of claim 1 , wherein the promoter is a constitutive promoter.

7. The production strain bacterial cell of claim 1 , wherein the promoter is repressible.

8. The production strain bacterial cell of claim 1 , wherein the promoter has a strength that is greater than the Anderson Score strength of promoter BBa_J23114.

9. The production strain bacterial cell of claim 1 , wherein the nucleic acid vector further comprises an origin of replication that is operable in the target bacterial host cell.

10. The production strain bacterial cell of claim 1 , wherein the nucleic acid vector is devoid of a Cas adaption module.

11. The production strain bacterial cell of claim 1 , wherein the nucleic acid vector is devoid of a nucleotide sequence encoding one or more of a Cas1, Cas2, Cas4, Cas6, Cas7, and Cas8.

12. The production strain bacterial cell of claim 1 , wherein the second nucleotide sequence encodes one or more of (a)-(g):

(a) Cas11, Cas7, and Cas8a1;

(b) Cas8b1, Cas7, and Cas5;

(c) Cas5, Cas8c, and Cas7;

(d) Cas8U2, Cas7, Cas5, and Cas6;

(e) Cas10d, Cas7, and Cas5;

(f) Cas8e, Cas11, Cas7, Cas5, and Cas6; and

(g) Cas8f, Cas5, Cas7, and Cas6f.

13. The production strain bacterial cell of claim 12 , wherein the Type I Cas3 is a Cas3′ or Cas3″.

14. The production strain bacterial cell of claim 12 , wherein the Type I Cas3 is a Cas3, Cas3′ or Cas3″, and wherein the Type I Cas3 is between the promoter and the second nucleotide sequence.

15. The production strain bacterial cell of claim 14 , wherein the nucleic acid vector is devoid of a nucleotide sequence encoding a further Cas between the promoter and the Type I Cas3.

16. The production strain bacterial cell of claim 12 , wherein the vector comprises the CRISPR array, the CRISPR array is cognate with the Type I Cas3, and wherein:

(a) the CRISPR array is a Type IA array and the nucleic acid vector comprises Cas11, Cas7, and Cas8a1;

(b) the CRISPR array is a Type IB array and the nucleic acid vector comprises Cas8b1, Cas7, and Cas5;

(c) the CRISPR array is a Type IC array and the nucleic acid vector comprises Cas5, Cas8c, and Cas7;

(d) the CRISPR array is a Type IU array and the nucleic acid vector comprises Cas8U2, Cas7, Cas5, and Cas6;

(e) the CRISPR array is a Type ID array and the nucleic acid vector comprises Cas10d, Cas7, and Cas5;

(f) the CRISPR array is a Type IE array and the nucleic acid vector comprises Cas8e, Cas11, Cas7, Cas5, and Cas6; or

(g) the CRISPR array is a Type IF array and the nucleic acid vector comprises Cas8f, Cas5, Cas7, and Cas6f.

17. The production strain bacterial cell of claim 1 , wherein the Type I Cas3 and Cascade are:

(a) Type IA Cas and Cascade proteins;

(b) Type IB Cas and Cascade proteins;

(c) Type IC Cas and Cascade proteins;

(d) Type ID Cas and Cascade proteins;

(e) Type IE Cas and Cascade proteins;

(f) Type IF Cas and Cascade proteins; or

(g) Type IU Cas and Cascade proteins.

18. The production strain bacterial cell of claim 1 , wherein the Type I Cas3 and Cascade are E. coli Cas and Cascade proteins.

19. The production strain bacterial cell of claim 1 , wherein the Type I Cas3 and Cascade are E. coli, C. difficile, P. aeruginosa, K. pneumoniae, P. furiosus , or B. halodurans Cas and Cascade proteins.

20. The production strain bacterial cell of claim 1 , wherein the Type I Cas3 is a Cas3 of a CRISPR/Cas locus of E. coli , and wherein the distance between the Cas3-encoding sequence of the locus and its cognate promoter in E. coli is further than the distance between the Cas3-encoding sequence and the promoter for controlling the expression of Type I Cas3 in the nucleic acid vector.

21. The production strain bacterial cell of claim 1 , wherein the CRISPR array or the gRNA-encoding sequence is under the control of a second promoter that is different from the promoter that controls the expression of the Type I Cas3.

22. The production strain bacterial cell of claim 1 , wherein the nucleic acid vector is a plasmid or phagemid.

23. The production strain bacterial cell of claim 1 , wherein the production strain bacterial cell comprises a nucleotide sequence whose expression is inducible to produce phage coat proteins in the cell of the production strain,

wherein the production strain bacterial cell comprises amplified copies of the nucleic acid vector,

wherein the production strain bacterial cell is capable of packaging the amplified copies of the nucleic acid vector into phage particles or non-self-replicative transduction particles for introducing the amplified copies of the nucleic acid vector into the target host cell.

24. The production strain bacterial cell of claim 23 , wherein the nucleic acid vector is a plasmid or phagemid and the delivery vehicle is a non-replicative transduction particle.

25. The production strain bacterial cell of claim 1 , wherein the second nucleotide sequence is under the control of the same promoter as the first nucleotide sequence.

26. The production strain bacterial cell of claim 1 , wherein the target sequence of the target bacterial host cell is a chromosomal sequence of the target bacterial host cell.

27. The production strain bacterial cell of claim 1 , wherein the production strain bacterial cell is an Escherichia coli ( E. coli ) cell.

Assignments (1)

Priority Claims (2)

Continuity (2)

References Cited (400)