US 12,091,656 B2
Method for generating higher order genome editing libraries
Manuel Kaulich, Frankfurt am Main (DE); Andreas Ernst, Frankfurt am Main (DE); Martin Wegner, Bad Nauheim (DE); Valentina Diehl, Frankfurt am Main (DE); Rahel De Bruyn, Wuerzburg (DE); and Svenja Wiechmann, Frankfurt am Main (DE)
Assigned to Johann Wolfgang Goethe-Universität Frankfur, (DE)
Appl. No. 16/472,645
Filed by Johann Wolfgang Goethe-Universität Frankfurt am Main, Frankfurt am Main (DE)
PCT Filed Dec. 27, 2017, PCT No. PCT/EP2017/084625
§ 371(c)(1), (2) Date Jun. 21, 2019,
PCT Pub. No. WO2018/122248, PCT Pub. Date Jul. 5, 2018.
Claims priority of application No. 102016125894.6 (DE), filed on Dec. 29, 2016.
Prior Publication US 2019/0330616 A1, Oct. 31, 2019
Int. Cl. C12N 15/10 (2006.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01); C40B 20/04 (2006.01); C40B 30/04 (2006.01); C40B 40/02 (2006.01); C40B 40/06 (2006.01)
CPC C12N 15/102 (2013.01) [C07H 21/02 (2013.01); C07H 21/04 (2013.01); C12N 15/1037 (2013.01); C12N 15/1072 (2013.01); C40B 20/04 (2013.01); C40B 30/04 (2013.01); C40B 40/02 (2013.01); C40B 40/06 (2013.01); C12Y 207/07007 (2013.01); C12Y 302/02027 (2013.01); C12Y 605/01001 (2013.01)] 19 Claims
 
1. A method for generating a covalently closed circularized (ccc) DNA based small RNA expression vector or vector library, the method comprising the steps of:
(a) providing a single stranded (ss) phagemid vector comprising (i) at least one small RNA expression cassette comprising a RNA promoter and an empty target-small-RNA-sequence-introduction-site or a small RNA coding sequence, or partial sequence thereof, (ii) at least one fl-origin for replication (ORI) of single strand DNA,
(b) providing at least one species of mutagenic RNA or DNA-Primer, wherein the mutagenic RNA or DNA-primer has the following structure in 3′ to 5′ direction: a first homology region, a target sequence region encoding for a small RNA to be expressed, and a second homology region, wherein the first homology region is complementary to, or is capable of annealing to, a sequence of the ss-phagemid vector construct flanking the empty target-small-RNA-sequence-introduction-site or the small RNA or DNA coding sequence, or partial sequence thereof, on the 5′ side, and wherein the second homology region is complementary to, or is capable of annealing to, a sequence of the ss-phagemid vector construct flanking the empty target-small-RNA-sequence-introduction-site or the small RNA coding sequence, or partial sequence thereof, on the 3′ side,
(c) annealing of at least one species of mutagenic RNA or DNA-primer to the ss-phagemid vector construct and amplifying a covalently closed circularized (ccc)-heteroduplex dsDNA therefrom, and
(d) removing residual wild type phagemid vector DNA,
wherein the small RNA is a siRNA, shRNA, an anti-miR, or a guide RNA (gRNA).