US 12,286,624 B2
Method for generating a gene editing vector with fixed guide RNA pairs
Manuel Kaulich, Frankfurt am Main (DE); Ivan Dikic, Frankfurt am Main (DE); Martin Wegner, Bad Nauheim (DE); Yves Matthess, Fulda (DE); and Koraljka Husnjak, Frankfurt am Main (DE)
Assigned to JOHANN WOLFGANG GOETHE-UNIVERSITÄT FRANKFURT AM MAIN, Frankfurt (DE)
Appl. No. 16/973,011
Filed by Johann Wolfgang Goethe-Universität Frankfurt am Main, Frankfurt am Main (DE)
PCT Filed Jun. 11, 2019, PCT No. PCT/EP2019/065167
§ 371(c)(1), (2) Date Dec. 7, 2020,
PCT Pub. No. WO2019/234258, PCT Pub. Date Dec. 12, 2019.
Claims priority of application No. 18176677 (EP), filed on Jun. 8, 2018.
Prior Publication US 2021/0261954 A1, Aug. 26, 2021
Int. Cl. C12N 15/11 (2006.01)
CPC C12N 15/11 (2013.01) [C12N 2310/20 (2017.05); C12N 2330/51 (2013.01)] 10 Claims
 
1. A method for generating a covalently closed circularized (ccc) DNA vector for expressing a fixed pair of guide RNAs (gRNAs) comprising the steps of:
(a) Providing an enhanced recipient vector comprising:
(x) two inverted or extraverted enhanced gRNA expression cassettes, wherein each enhanced gRNA expression cassette comprises in that order: (i) optionally an RNA promoter, (ii) a gRNA placeholder sequence, and (iii) optionally a crRNA sequence; and
(y) a tracrRNA expression cassette;
(b) Providing an enhanced mutagenic DNA primer comprising two gRNA coding sequences of interest and homology regions capable to mediate a binding of the mutagenic DNA primer to the two inverted or extraverted enhanced gRNA expression cassettes; and
(c) Generating a cccDNA vector using the recipient vector and the mutagenic DNA primer.