US 12,378,546 B2
Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption
Andrew M. Scharenberg, Seattle, WA (US); Michael T. Certo, Seattle, WA (US); and Kamila Sabina Gwiazda, Seattle, WA (US)
Assigned to SEATTLE CHILDREN'S RESEARCH INSTITUTE, Seattle, WA (US)
Filed by SEATTLE CHILDREN'S HOSPITAL, Seattle, WA (US)
Filed on Dec. 1, 2023, as Appl. No. 18/526,930.
Application 14/949,744 is a division of application No. 14/173,705, filed on Feb. 5, 2014, abandoned.
Application 14/173,705 is a division of application No. 13/405,183, filed on Feb. 24, 2012, granted, now 8,673,557, issued on Mar. 18, 2014.
Application 18/526,930 is a continuation of application No. 17/244,190, filed on Apr. 29, 2021, granted, now 11,873,479.
Application 17/244,190 is a continuation of application No. 15/215,405, filed on Jul. 20, 2016, granted, now 11,008,565, issued on May 18, 2021.
Application 15/215,405 is a continuation of application No. 14/949,744, filed on Nov. 23, 2015, granted, now 10,995,332, issued on May 4, 2021.
Claims priority of provisional application 61/447,672, filed on Feb. 28, 2011.
Prior Publication US 2024/0102001 A1, Mar. 28, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); A61K 9/00 (2006.01); A61K 35/28 (2015.01); A61K 38/45 (2006.01); A61K 38/46 (2006.01); A61K 38/52 (2006.01); C12N 9/12 (2006.01); C12N 9/14 (2006.01); C12N 9/90 (2006.01); C12N 15/10 (2006.01); C12N 15/62 (2006.01); C12Q 1/00 (2006.01); C12Q 1/34 (2006.01); A61K 35/12 (2015.01)
CPC C12N 15/102 (2013.01) [A61K 9/0019 (2013.01); A61K 35/28 (2013.01); A61K 38/45 (2013.01); A61K 38/465 (2013.01); A61K 38/52 (2013.01); C12N 9/1252 (2013.01); C12N 9/22 (2013.01); C12N 9/90 (2013.01); C12N 15/62 (2013.01); A61K 2035/124 (2013.01); C07K 2319/60 (2013.01); C12N 2800/80 (2013.01); C12N 2840/203 (2013.01)] 17 Claims
 
1. A method of increasing mutagenesis at a double-strand DNA (dsDNA) break at a selected dsDNA target site in a eukaryotic cell comprising:
a) selecting a dsDNA target site for mutagenesis; and
b) introducing into the eukaryotic cell a polynucleotide sequence encoding:
(i) an endonuclease having a >14 base pair cleavage site, wherein said endonuclease binds and cleaves the selected dsDNA target site; and
(ii) an exonuclease;
wherein the exonuclease exhibits exonuclease activity at the cleaved dsDNA target site, resulting in increased mutagenesis at the selected dsDNA target site as compared to mutagenesis that occurs in the absence of exonuclease activity.