Compositions and methods using Cas9 with enhanced spacer acquisition function

Patent metadata

Field	Value
Publication number	US-11377645-B2
Application number	US-201716470195-A
Country	US
Kind code	B2
Filing date	Dec 18, 2017
Priority date	Dec 16, 2016
Publication date	Jul 5, 2022
Grant date	Jul 5, 2022

Abstract

Provided are Cas9 enzymes that have mutations that enhance their properties, relative to un-mutated Cas9. The altered Cas9 enzymes exhibit i) an increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG PAMs, or a combination of i) and ii). The altered Cas9 enzymes comprise an amino acid substitution of 1473 and K500 in a Streptococcus pyogenes or similar Cas9 enzyme. Also provided are polynucleotides, including expression vectors that encode the Cas9 enzymes, cells that contain the polynucleotides, and methods of making and using such cells. The disclosure includes tagging, or labelling bacteria, and for enhancing phage acquired immunity in bacteria, such as those used in industrial processes, including the food and beverage industry, such as the dairy industry. The food products are also included.

First claim

What is claimed is:

1. An expression vector encoding a Cas9 enzyme comprising a substitution, wherein the substitution is of at least one of the following amino acids: I473 and K500 in SEQ ID NO:1, and wherein relative to a control the Cas9 enzyme exhibits at least one of: i) increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG protospacer adjacent motifs (PAMs), wherein the control comprises a rate of spacer acquisition, or increased cleavage efficiency of targets with NAG PAMs, produced by a Cas9 enzyme comprising the sequence of SEQ ID NO:1.

2. The expression vector of claim 1 , wherein the Cas9 enzyme is a Streptococcus pyogenes Cas9 enzyme.

3. The expression vector of claim 1 , wherein the substitution comprises I473F.

4. The expression vector of claim 1 , wherein the substitution comprises I473F and K500I.

5. Bacteria comprising an expression vector of claim 1 .

6. A method of making modified bacteria comprising introducing into the bacteria an expression vector of claim 4 .

7. A method comprising contacting bacteria of claim 5 with one or more bacteriophage such that at least one spacer sequence in the genome of the bacteriophage is acquired by the bacteria.

8. The method of claim 7 , wherein the bacteria are contacted with a plurality of distinct bacteriophage, and wherein the bacteria acquire a plurality of distinct spacer sequences from the plurality of the bacteriophage, and wherein the number of distinct spacers in the plurality is greater that a control value, and/or the distinct spacers in the plurality are acquired more quickly than for a control value, and wherein the control value comprises a number of distinct spacers, or a rate at which spacers are acquired, obtained by contacting bacteria that express a Cas9 enzyme comprising the sequence of SEQ ID NO:1 with a plurality of distinct bacteriophage.

9. The method of claim 7 , wherein the bacteriophage are obtained from a bacterial culture used to produce a food product or a beverage.

10. The method of claim 9 , wherein the food product comprises a dairy product.

11. A food product comprising bacteria of claim 5 .

12. The food product of claim 11 , wherein the food product comprises a dairy product.

13. A method for labeling bacteria with one or more spacer sequences, the method comprising introducing into the bacteria an expression vector of claim 1 , and introducing into the bacteria a polynucleotide comprising at least one spacer sequence.

14. The method of claim 13 , further comprising determining the sequence of at least one spacer sequence from bacteria that acquired the spacer sequence.

15. A Cas9 enzyme comprising an amino acid substitution, wherein the amino acid substitution is of at least one of the following amino acids: I473 and K500 in SEQ ID NO:1, and wherein relative to a control the Cas9 enzyme exhibits at least one of: i) increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG protospacer adjacent motifs (PAMs), wherein the control comprises a rate of spacer acquisition, or increased cleavage efficiency of targets with NAG PAMs, produced by a Cas9 enzyme comprising the sequence of SEQ ID NO:1.

16. The Cas9 enzyme of claim 15 , wherein the Cas9 enzyme is a Streptococcus pyogenes Cas9 enzyme.

Assignees

• Univ Rockefeller

Inventors

• Marraffini Luciano
• Heller Robert
• Bikard David

Classifications

• C12G1/00

  Preparation of wine or sparkling wine · CPC title

• C12N2820/55

  from bacteria · CPC title

• A23C9/123

  using only microorganisms of the genus lactobacteriaceae; Yoghurt (A23C9/13 takes precedence) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title