US 12,123,033 B2
Modified double-stranded donor templates
Jessica Woodley, North Liberty, IA (US); Bernice Thommandru, Iowa City, IA (US); Joseph Dobosy, Coralville, IA (US); Mark Behlke, Coralville, IA (US); Adam Clore, Iowa City, IA (US); Garrett Rettig, Coralville, IA (US); and Beimeng Sun, North Liberty, IA (US)
Assigned to INTEGRATED DNA TECHNOLOGIES, INC., Coralville, IA (US)
Filed by INTEGRATED DNA TECHNOLOGIES, INC., Coralville, IA (US)
Filed on Oct. 23, 2020, as Appl. No. 17/079,097.
Claims priority of provisional application 62/925,366, filed on Oct. 24, 2019.
Prior Publication US 2021/0123035 A1, Apr. 29, 2021
Int. Cl. C12N 15/66 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01)
CPC C12N 9/22 (2013.01) [C12N 15/113 (2013.01); C12N 15/66 (2013.01); C12N 2310/122 (2013.01); C12N 2310/20 (2017.05); C12N 2310/321 (2013.01); C12N 2310/531 (2013.01); C12N 2800/80 (2013.01)] 7 Claims
 
1. A double stranded DNA homology directed repair (HDR) donor comprising:
a first homology arm region,
an insert region,
a second homology arm region; and
universal primer sequences terminally flanking the first homology arm region and the second homology arm region;
wherein:
the first homology arm region and the second homology arm region comprise one or more 2′-OME, 2′-MOE, 2′-F, or 2′-oxygen-4′-carbon methylene (Locked Nucleic Acid) modifications of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the first homology arm region and the second homology arm region;
the 2′-OME and 2′-MOE modifications improve homology directed repair efficiency and reduce homology-independent integration; and
the Locked Nucleic Acid and 2′-F modifications improve homology directed repair efficiency and increase homology-independent integration.