US 12,221,606 B2
Using nucleosome interacting protein domains to enhance targeted genome modification
Fuqiang Chen, St. Louis, MO (US); Xiao Ding, St. Louis, MO (US); Yongmei Feng, St. Louis, MO (US); and Gregory Davis, Webster Groves, MO (US)
Assigned to Sigma-Aldrich Co. LLC, St. Louis, MO (US)
Filed by Sigma-Aldrich Co. LLC, St. Louis, MO (US)
Filed on Feb. 18, 2020, as Appl. No. 16/793,253.
Application 16/793,253 is a division of application No. 16/031,819, filed on Jul. 10, 2018, granted, now 10,604,752.
Claims priority of provisional application 62/531,222, filed on Jul. 11, 2017.
Prior Publication US 2020/0208135 A1, Jul. 2, 2020
Int. Cl. C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12N 15/85 (2006.01); C12N 15/90 (2006.01); C12Q 1/6897 (2018.01); A61K 48/00 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/85 (2013.01); C12N 15/907 (2013.01); C12Q 1/6897 (2013.01); A61K 48/005 (2013.01); C07K 2319/09 (2013.01); C07K 2319/10 (2013.01); C07K 2319/80 (2013.01); C07K 2319/81 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 13 Claims
 
1. A method for increasing cleavage efficiency of targeted genome modification or increasing epigenetic modification in a eukaryotic cell, the method comprising introducing into the eukaryotic cell:
(a) at least one fusion protein or nucleic acid encoding at least one fusion protein, each fusion protein comprising a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein (i) has nuclease or nickase activity or (ii) is modified to lack all nuclease activity and is linked to a non-nuclease domain; and
(b) at least one guide RNA or nucleic acid encoding at least one guide RNA; wherein the CRISPR protein of the at least one fusion protein is targeted to a target chromosomal sequence and the at least one nucleosome interacting protein domain of the at least one fusion protein alters nucleosomal or chromatin structure such that the at least one fusion protein has increased access to the target chromosomal sequence, thereby increasing cleavage efficiency of targeted genome modification or increasing epigenetic modification, wherein the at least one nucleosome interacting protein domain is a high mobility group (HMG) box (HMGB) DNA binding domain, a HMG nucleosome-binding (HMGN) protein, a central globular domain from a histone H1 variant comprising SEQ ID NO: 45, or a combination thereof, wherein the CRISPR protein is a type II CRISPR/Cas9 protein or a type V-A CRISPR/Cpf1 protein.