US 12,337,291 B2
Guide RNA synthesis system and method
Paul Dabrowski, Atherton, CA (US); Fabian Gerlinghaus, San Francisco, CA (US); Reed Kelso, San Francisco, CA (US); and John Andrew Walker, II, San Leandro, CA (US)
Assigned to Synthego Corporation, Redwood City, CA (US)
Filed by Synthego Corporation, Redwood City, CA (US)
Filed on Aug. 10, 2020, as Appl. No. 16/989,593.
Application 16/989,593 is a continuation of application No. 16/130,901, filed on Sep. 13, 2018, abandoned.
Application 16/130,901 is a continuation of application No. PCT/US2018/050306, filed on Sep. 10, 2018.
Claims priority of provisional application 62/556,791, filed on Sep. 11, 2017.
Prior Publication US 2021/0047668 A1, Feb. 18, 2021
Int. Cl. C12P 19/34 (2006.01); B01J 19/00 (2006.01); B01L 7/00 (2006.01); C07H 1/00 (2006.01); C07K 1/04 (2006.01); C12N 15/10 (2006.01); C40B 50/14 (2006.01)
CPC B01J 19/0046 (2013.01) [B01L 7/52 (2013.01); C07H 1/00 (2013.01); C07K 1/045 (2013.01); C07K 1/047 (2013.01); C12N 15/10 (2013.01); C12N 15/1093 (2013.01); C40B 50/14 (2013.01); B01J 2219/00306 (2013.01); B01J 2219/00351 (2013.01); B01J 2219/00418 (2013.01); B01J 2219/00423 (2013.01); B01J 2219/00454 (2013.01); B01J 2219/00495 (2013.01); B01J 2219/00596 (2013.01); B01J 2219/00695 (2013.01); B01J 2219/00722 (2013.01); B01J 2219/00725 (2013.01)] 15 Claims
 
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.