US 11,866,726 B2
Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
Cecilia Cotta-Ramusino, Cambridge, MA (US); and Carrie M. Margulies, Waban, MA (US)
Assigned to Editas Medicine, Inc., Cambridge, MA (US)
Appl. No. 16/630,510
Filed by Editas Medicine, Inc., Cambridge, MA (US)
PCT Filed Jul. 13, 2018, PCT No. PCT/US2018/042040
§ 371(c)(1), (2) Date Jan. 13, 2020,
PCT Pub. No. WO2019/014564, PCT Pub. Date Jan. 17, 2019.
Claims priority of provisional application 62/582,563, filed on Nov. 7, 2017.
Claims priority of provisional application 62/532,509, filed on Jul. 14, 2017.
Prior Publication US 2020/0165636 A1, May 28, 2020
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 5/0735 (2010.01); C12N 5/0789 (2010.01); C12N 5/074 (2010.01); C12N 15/113 (2010.01); C12N 15/86 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 5/0606 (2013.01); C12N 5/0647 (2013.01); C12N 5/0696 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/113 (2013.01); C12N 15/86 (2013.01); C12N 2310/20 (2017.05)] 12 Claims
 
1. An isolated nucleic acid for homologous recombination with a target nucleic acid having a cleavage site, wherein:
(a) a first strand of the target nucleic acid comprises, from 5′ to 3′, P1-H1-X-H2-P2, wherein
P1 is a first priming site;
H1 is a first homology arm;
X is the cleavage site;
H2 is a second homology arm; and
P2 is a second priming site; and
(b) a first strand of the isolated nucleic acid comprises, from 5′ to 3′, A1-S1-P2′-N-P1′-S2-A2, wherein
A1 is a homology arm that is substantially identical to H1;
P2′ is a priming site that is substantially identical to P2;
N is a cargo;
P1′ is a priming site that is substantially identical to P1;
A2 is a homology arm that is substantially identical to H2;
wherein S1 is a first stuffer, wherein S2 is a second stuffer, and wherein each of S1 and S2 comprise a random or heterologous sequence having a GC content of approximately 40%.