Compositions and methods of use for small-molecule regulation of CRISPR-Cas9 activity using RNA aptamers

Patent metadata

Field	Value
Publication number	US-12416008-B2
Application number	US-202117375808-A
Country	US
Kind code	B2
Filing date	Jul 14, 2021
Priority date	Jan 17, 2019
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

Abstract

Provided herein are single guide RNAs (sgRNAs) that comprise aptamer sequences and related compositions and methods. Also provided herein are methods of selecting inducible sgRNAs that comprise aptamer sequences.

First claim

What is claimed is:

1. A guide RNA (sgRNA) comprising a small-molecule-binding aptamer sequence adjacent to a region comprising a 2×4 internal loop and a region comprising an upper stem, wherein the small-molecule-binding aptamer is a theophylline-binding aptamer and the theophylline-binding aptamer comprises at least 85% homology to a polynucleotide represented by the sequence 5′-AUACCAGCUUCGAAAGAAGCCCUUGGCAG-3′ (SEQ ID NO: 95).

2. The sgRNA of claim 1 , wherein sgRNA comprises up to 2 nucleotides between the small-molecule-binding aptamer sequence and the 2×4 internal loop.

3. A ribonucleoprotein (RNP) comprising an sgRNA of claim 1 and a Cas enzyme.

4. The RNP of claim 3 , wherein the Cas enzyme is Cas9.

5. The RNP of claim 3 , wherein the region comprising the 2×4 internal loop and the region comprising the upper stem comprises the sequence of SEQ ID NO: 169.

6. The RNP of claim 3 , wherein the region comprising the 2×4 internal loop and the region comprising the upper stem comprises the sequence of SEQ ID NO: 100.

7. A method of inducing gene editing in a bacterium, the method comprising:
a. introducing or expressing the sgRNA of claim 1 in a bacterium,
b. introducing or expressing a Cas enzyme in the bacterium, and
c. contacting the bacterium with a small molecule that interacts with the small-molecule-binding aptamer sequence in the sgRNA such that gene editing is induced in the bacterium.

8. The method of claim 7 , wherein the Cas enzyme is Cas9.

9. The method of claim 7 , wherein the sgRNA exhibits greater than or equal to 10-fold induction in editing in presence of a small molecule ligand that binds to the small-molecule-binding aptamer sequence.

10. An in vivo method of selecting in bacteria a switchable aptamer single guide RNA (agRNA) that is inducible with a small molecule, wherein the switchable agRNA comprises a single guide RNA of claim 1 , the method comprising:
a. performing a negative selection step comprising growing a first plurality of bacteria in absence of the small molecule and in the absence of X-red protein expression, inducing cell death in a subset of the plurality of bacteria to produce a second plurality of bacteria, wherein individual bacteria in the first plurality of bacteria comprise a nucleic acid encoding the candidate agRNA comprising the sgRNA of claim 1 that produces a cut site in a gene encoding a selection marker protein: a Cas enzyme; and a template for homologous repair of the cut site in the gene encoding the selection marker protein;
b. performing a positive selection step comprising either:
i. growing the second plurality of bacteria in the presence of the small molecule and red protein expression; or
ii. isolating nucleic acids encoding the candidate agRNAs comprising the sgRNA of claim 1 from the second plurality of bacteria and introducing the nucleic acids into a third plurality of bacteria and growing the third plurality of bacteria in the presence of the small molecule and X-red protein expression, wherein individual bacteria in the third plurality comprise a nucleic acid encoding a candidate agRNA that produces a cut site in the gene encoding a selection marker protein, a Cas enzyme, and a template for homologous repair of the cut site in the gene that encodes the selection marker protein; and
c. selecting at least one bacterium from b. that expresses an altered selection marker protein.

11. The method of claim 10 , wherein the selection marker protein is galK and the template for homologous repair comprises a premature stop codon for a galK gene.

12. An in vivo method of selecting in bacteria a switchable aptamer single guide RNA (agRNA) that is inducible with a small molecule, wherein the switchable agRNA comprises a single guide RNA of claim 1 , the method comprising:
a. performing a negative selection step comprising growing a first plurality of bacteria in absence of the small molecule and in presence of X-red protein expression and inducing cell death in a subset of a first plurality of bacteria to produce a second plurality of bacteria, wherein individual bacteria in the first plurality of bacteria comprise a nucleic acid encoding the candidate agRNA comprising the sgRNA of claim 1 that produces a cut site in a gene encoding a selection marker protein, a Cas enzyme, and a template for homologous repair of the cut site in the gene that encodes the selection marker protein;
b. performing a positive selection step comprising either:
i. growing the second plurality of bacteria in the presence of the small molecule and X-red protein expression; or
ii. isolating nucleic acids encoding candidate agRNAs from the second plurality of bacteria and introducing the nucleic acids into a third plurality of bacteria and growing the third plurality of bacteria in the presence of the small molecule and λ-red protein expression, wherein individual bacteria in the third plurality comprise a nucleic acid encoding the candidate agRNAs comprising the sgRNA of claim 1 that produces a cut site in the gene encoding a selection marker protein, a Cas enzyme, and a template for homologous repair of the cut site in the gene that encodes the selection marker protein; and
c. selecting at least one bacterium from b. that expresses the selection marker protein.

13. The sgRNA of claim 1 , wherein the region comprising the 2×4 internal loop and the upper stem comprises the sequence of SEQ ID NO: 169.

14. A composition comprising: (i) the sgRNA of claim 1 ; or (ii) a vector expressing the sgRNA of claim 1 .

15. The sgRNA of claim 1 , wherein the small-molecule-binding aptamer is a theophylline-binding aptamer, and the theophylline-binding aptamer comprises the polynucleotide represented by the sequence 5′-AUACCAGCUUCGAAAGAAGCCCUUGGCAG-3′ (SEQ ID NO: 95).

16. A guide RNA (sgRNA) comprising a small-molecule-binding aptamer sequence adjacent to a region comprising a 2×4 internal loop and a region comprising an upper stem, wherein the region comprising the 2×4 internal loop and the upper stem comprises the sequence 5′-GNNNGGAUACCAGCUUCGAAAGAAGCCNUUGGCAGCCNNNNNC-3′ (SEQ ID NO: 169).

17. The sgRNA of claim 16 , wherein sgRNA comprises up to 2 nucleotides between the small-molecule-binding aptamer sequence and the 2×4 internal loop.

18. A guide RNA (sgRNA) comprising a small-molecule-binding aptamer sequence adjacent to a region comprising a 2×4 internal loop and a region comprising an upper stem, wherein the region comprising the 2×4 internal loop and the upper stem comprises a polynucleotide represented by SEQ ID NO: 100, UGAAGGAUACCAGCUUCGAAAGAAGCCNUUGGCAGCCGCAACA.

19. The sgRNA of claim 18 , wherein sgRNA comprises up to 2 nucleotides between the small-molecule-binding aptamer sequence and the 2×4 internal loop.

Assignees

• Univ Colorado Regents
• Inscripta Inc

Inventors

• Batey Robert
• Iwasaki Cordero Roman S
• Garst Andrew

Classifications

• C12N2310/16

  Aptamers · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

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

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title