Granted Patent B2

US 12,337,291 · App. 16/989,593 · Granted Jun 24, 2025

Inventors: Paul Dabrowski (Atherton, CA); Fabian Gerlinghaus (San Francisco, CA); Reed Kelso (San Francisco, CA); John Andrew Walker, II (San Leandro, CA)

Assignee: Synthego Corporation

B01J19/0046B01L7/52C07H1/00C07K1/045C07K1/047C12N15/10C12N15/1093C40B50/14B01J2219/00306B01J2219/00351B01J2219/00418B01J2219/00423B01J2219/00454B01J2219/00495B01J2219/00596B01J2219/00695B01J2219/00722B01J2219/00725

Abstract

Claims (22)

1. A method of synthesizing a plurality of single molecule guide RNAs (sgRNAs) comprising identical scaffold sequences and multiple different protospacer domains targeting distinct genomic regions, the method comprising:

(a) synthesizing a plurality of identical scaffold sequences onto a plurality of solid supports in a single vessel of a first oligosynthesizer;

wherein each of the plurality of identical scaffold sequences consists of a fusion of a transactivating clustered regularly interspaced palindromic repeat RNA (tracrRNA) sequence and a clustered regularly interspaced palindromic repeat RNA (crRNA) repeat sequence joined by a loop sequence to form a plurality of identical scaffold sequences, such that each of the plurality of solid supports comprises one or more of the plurality of identical scaffold sequences covalently bound to the 3′ end of the tracrRNA sequence;

(b) splitting the plurality of solid supports into a plurality of separate reaction vessels; and

(c) coupling, in a different oligonucleotide synthesizer, a plurality of different protospacer domains to the 5′ end of the plurality of identical scaffold sequences bound to the plurality of solid supports within each of the plurality of separate reaction vessels to synthesize a plurality of different single guide RNAs (sgRNAs), such that after the coupling each separate reaction vessel comprises a plurality of identical sgRNAs,

wherein each of the plurality of different protospacer domains is specific to a DNA target site, and

wherein the coupling of each of the plurality of different protospacer domains comprises phosphoramidite coupling of nucleotide monomers starting from the 5′ end of each of the plurality of identical scaffold sequences and/or phosphoramidite coupling of different oligonucleotides; and

(d) cleaving the plurality of synthesized sgRNAs from the plurality of identical scaffold sequences, wherein each of the plurality of synthesized sgRNAs is ˜100 nucleotides (nt) in length.

2. The method of claim 1 , wherein the plurality of solid supports comprise a polystyrene resin.

3. The method of claim 1 , wherein the nucleotide monomers are selected from the group consisting of an adenine deoxyribonucleoside phosphoramidite, a thymine deoxyribonucleoside phosphoramidite, a cytosine deoxyribonucleoside phosphoramidite, a guanine deoxyribonucleoside phosphoramidite, and a combination thereof.

4. The method of claim 1 , wherein the each of the plurality of identical scaffold sequences comprises an RNA polynucleotide about 80 nucleotides in length.

5. The method of claim 1 , wherein each of the plurality of different protospacer domains of the sgRNAs is about 17 to 22 nucleotides in length.

6. The method of claim 1 , further comprising adding ammonium hydroxide to the plurality of separate reaction vessels to cleave one or more of the plurality of synthesized sgRNAs from the solid support.

7. The method of claim 6 , further comprising lyophilizing the plurality of synthesized sgRNAs.

8. The method of claim 7 , further comprising desalting the plurality of synthesized sgRNAs.

9. The method of claim 7 , wherein purifying the plurality of synthesized sgRNAs comprises reverse phase high performance liquid chromatography.

10. The method of claim 9 , wherein purifying the plurality of synthesized sgRNAs comprises reverse phase high performance liquid chromatography.

11. The method of claim 1 , further comprising: acquiring a sample of the plurality of solid supports from the first oligosynthesizer after step (a) and before splitting the plurality of solid supports into the plurality of separate reaction vessels in step (b), assessing a quantity of the plurality of identical scaffold sequences bound to the plurality of solid supports within the acquired sample; and prior to step (c), determining the quantity of the plurality of identical scaffold sequences synthesized in the first oligosynthesizer within the single vessel based on an assessed quantity of the plurality of identical scaffold sequences in the acquired sample.

12. The method of claim 11 , further comprising: determining the quantity of the plurality of solid supports to be split into the plurality of separate reaction vessels in step (b) based on an assessed quantity of the plurality of identical scaffold sequences within the acquired sample.

13. The method of claim 12 , further comprising: cleaving the plurality of identical scaffold sequences from the plurality of solid supports within the sample before assessing the quantity of the plurality of identical scaffold sequences within the acquired sample.

14. The method of claim 1 , wherein the nucleotide monomers are selected from the group consisting of a 2′-O-thionocarbamate protected adenine nucleoside phosphoramidite, a 2′-O-thionocarbamate-protected uracil nucleoside phosphoramidite, a 2′-O-thionocarbamate-protected cytosine nucleoside phosphoramidite, a 2′-O-thionocarbamate-protected guanine nucleoside phosphoramidite, and a combination thereof.

15. The method of claim 1 , wherein each of the plurality of separate reaction vessels comprises a different protospacer domain.

Assignments (2)

Continuity (4)

References Cited (281)