Production of unnatural nucleotides using a CRISPR/Cas9 system
Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.
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.