US 11,905,521 B2
Methods and systems for targeted gene manipulation
Bo Feng, Shatin (CN); and Xiangjun He, Shatin (CN)
Assigned to The Chinese University of Hong Kong, Shatin (CN)
Filed by THE CHINESE UNIVERSITY OF HONG KONG, Shatin (CN)
Filed on Nov. 17, 2016, as Appl. No. 15/354,329.
Claims priority of provisional application 62/288,974, filed on Jan. 29, 2016.
Claims priority of provisional application 62/256,514, filed on Nov. 17, 2015.
Prior Publication US 2017/0198302 A1, Jul. 13, 2017
Int. Cl. C12N 15/00 (2006.01); C12N 15/85 (2006.01); C12Q 1/6897 (2018.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01)
CPC C12N 15/85 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/1086 (2013.01); C12Q 1/6897 (2013.01)] 15 Claims
 
1. A composition comprising:
(1) a donor construct for gene integration into a target locus within a mammalian host cell genome, comprising (a) a coding sequence for at least one gene; (b) a polyA segment at the 3′ end of the at least one gene coding sequence; (c) an internal ribosome entry site (ires) at the 5′ end of the at least one gene coding sequence; (d) one copy of a first sgRNA target sequence located at the 5′ end of the ires, wherein the first sgRNA target sequence is about 20 to about 40 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 5 or its reverse complementary sequence; (e) tandem repeats of human insulator sequences located at the 5′ end of the first sgRNA target sequence site located at the 5′ end of the ires; and (f) a nucleotide sequence containing multiple stop codons inserted at the 5′ end of the ires element;
wherein the donor construct is a promoterless construct;
and wherein the donor construct does not comprise substantial sequence identity to the target locus within the mammalian host cell genome; and
(2) two sgRNAs targeting different target sequences and/or one or two DNA molecules encoding the two sgRNAs, wherein one of the sgRNAs targets the first sgRNA target sequence on the donor construct, and the other sgRNA targets a second sgRNA target sequence within a 3′ untranslated region (UTR) of the target locus within the mammalian host cell genome.