Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides
View Patent ↗Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
1. A method for producing a Cas nuclease/guide RNA complex, comprising:
(a) combining:
a DNA polymerase,
an RNA polymerase,
a first synthetic single stranded (ss) DNA oligonucleotide,
a second synthetic single stranded (ss) DNA oligonucleotide,
wherein the first ss DNA oligonucleotide comprises a promoter sequence for the polymerase and a sequence at the 3′ end that hybridizes to a complementary sequence in the second ss DNA oligonucleotide;
dNTPs,
rNTPS, and
a Cas nuclease, to produce a reaction mix; and
(b) incubating the reaction mix to produce a Cas nuclease/guide RNA complex that comprises a guide RNA that is at least partially transcribed from the second oligonucleotide.
2. The method according to claim 1 , further comprising adding a ligase and a third synthetic single stranded (ss) DNA oligonucleotide to the reaction mix before step (b), wherein the third ss DNA oligonucleotide comprises a sequence at the 5′ end that hybridizes to a complementary sequence in the second ss DNA oligonucleotide and the guide RNA is at least partially transcribed from the second oligonucleotide and the complement of the third oligonucleotide.
3. The method according to claim 1 , wherein the DNA polymerase is a strand displacing polymerase.
4. The method according to claim 1 , wherein the first ss DNA oligonucleotide contains a sequence module in which the sequence is variable.
5. The method according to claim 4 , wherein the variable sequence module is DNA targeting sequence for a Cas nuclease.
6. The method according to claim 1 , wherein the method comprises producing a plurality of said reaction mixes in a multiwell plate, wherein the sequence of the first ssDNA oligonucleotide differs in each reaction mix.
7. The method according to claim 6 , wherein the second ss DNA oligonucleotide is the same in each reaction.
8. The method according to claim 5 , wherein the second ss DNA oligonucleotide comprises a tracrRNA sequence.