Production of unnatural nucleotides using a CRISPR/Cas9 system

Patent metadata

Field	Value
Publication number	US-11761007-B2
Application number	US-201616063107-A
Country	US
Kind code	B2
Filing date	Dec 16, 2016
Priority date	Dec 18, 2015
Publication date	Sep 19, 2023
Grant date	Sep 19, 2023

Abstract

Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.

First claim

What is claimed is:

1. A cell comprising:
(I) a first nucleic acid sequence comprising an unnatural nucleotide, wherein the unnatural nucleotide does not substantially form a base pair with a natural nucleotide; and
(II) a second nucleic acid sequence encoding a CRISPR/Cas system, wherein the CRISPR/Cas system encodes a single guide RNA (sgRNA) comprising a crRNA-tracrRNA scaffold and a target motif that is complementary to a modified nucleic acid sequence, and
wherein the modified nucleic acid sequence is identical to the first nucleic acid sequence except that it comprises: (1) a substitution of the unnatural nucleotide with a natural nucleotide or (2) a single nucleotide deletion at the unnatural nucleotide position.

2. The cell of claim 1 , wherein one or more plasmids comprise the second nucleic acid sequence encoding the CRISPR/Cas system and the first nucleic acid sequence comprising the unnatural nucleotide.

3. The cell of claim 1 , wherein the modified nucleic acid comprises a substitution of the unnatural nucleotide with a natural nucleotide.

4. The cell of claim 1 , wherein the sgRNA further comprises a protospacer adjacent motif (PAM) recognition element.

5. The cell of claim 4 , wherein a nucleotide within the target motif that recognizes the substituted natural nucleotide of the modified nucleic acid sequence is located between 3 to 22, between 5 to 20, between 5 to 18, between 5 to 15, between 5 to 12, or between 5 to 10 nucleotides from the 5′ terminus of the PAM recognition element.

6. The cell of claim 1 , wherein the CRISPR/Cas system comprises a wild-type Cas9 polypeptide.

7. The cell of claim 1 , wherein the unnatural nucleotide comprises an unnatural base selected from

8. The cell of claim 1 , further comprising a third nucleic acid sequence that encodes an additional single guide RNA (sgRNA) comprising a crRNA-tracrRNA scaffold.

9. The cell of claim 1 , wherein the first nucleic acid sequence comprising the unnatural nucleotide further comprises an additional unnatural nucleotide.

10. The cell of claim 1 , wherein the cell is E. coli.

11. A cell comprising:
(I) a first nucleic acid sequence comprising an unnatural nucleotide, wherein the unnatural nucleotide does not substantially form a base pair with a natural nucleotide; and
(II) one or more second nucleic acid sequences encoding a CRISPR/Cas system, wherein the CRISPR/Cas system encodes two or more single guide RNAs (sgRNAs), each of which comprises a crRNA-tracrRNA scaffold and wherein each sgRNA comprises a target motif that is complementary to a modified nucleic acid sequence, and
wherein the modified nucleic acid sequence is identical to the first nucleic acid sequence except that it comprises: (1) a substitution of the unnatural nucleotide with a natural nucleotide or (2) a single nucleotide deletion at the unnatural nucleotide position.

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Lamb Brian
• Zhang Yorke

Classifications

• C12N9/22 Primary

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

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2310/20

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

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title