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US 12,606,844 B2Granted
Methods and systems for targeted gene manipulation
AssigneeThe Chinese University of Hong Kong
InventorsBo Feng, Xiangjun He, Zhenjie Zhang, Kwan Yuen Katherine Xue
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Abstract
The present invention provides novel methods and systems, including cell lines, recombinant polynucleotide constructs, compositions, and kits, for targeted yet universal genomic manipulation.
Claims17
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) two copies of a first sgRNA target sequence, one copy located at the 5′ end of the ires and the other copy located at the 3′ end of the polyA segment, 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.
7 more elements
An isolated mammalian host cell 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) two copies of a first sgRNA target sequence, one copy located at the 5′ end of the ires and the other copy located at the 3′ end of the polyA segment, 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′ UTR of the target locus within the mammalian host cell genome.
7 more elements
An isolated mammalian host cell comprising a donor construct for gene integration into a target locus within the mammalian host cell genome, the donor construct 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) two copies of a sgRNA target sequence, one copy located at the 5′ end of the ires and the other copy located at the 3′ end of the poly A segment, wherein the 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 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;
- wherein the donor construct does not comprise substantial sequence identity to the target locus within the mammalian host cell genome;
- and wherein the donor construct is integrated at a 3′UTR of the target locus within the mammalian host cell genome.
5 more elements
Dependencies are inferred from the claim text.