Nucleic acid constructs comprising gene editing multi-sites and uses thereof
AssigneeIO Bioscience, Inc.
InventorsSicco Hans Popma, Di Zhang
Life of this patent
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Abstract
Disclosed herein is a polynucleotide construct comprising one or more nuclease recognition sequences upstream and downstream of a Gene editing multi-site that comprises a plurality of nuclease recognition sequences. The plurality of nuclease recognition sequences facilitate insertion of one or more exogenous donor genes into the host cell.
Claims20
An isolated gene editing multi-site (GEMS) construct for insertion into a genome at an insertion site, wherein said GEMS construct comprises:
- a GEMS sequence that comprises a plurality of nuclease recognition sequences, wherein at least one of said plurality of nuclease recognition sequences is selected from the group consisting of sequences set forth in SEQ ID NOs:
- 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and reverse complements thereof.
An isolated genetically engineered cell that comprises a gene editing multi-site (GEMS) sequence in said cell's genome, wherein said GEMS sequence comprises a plurality of nuclease recognition sequences, wherein at least one of said plurality of nuclease recognition sequences is selected from the group consisting of sequences set forth in SEQ ID NOs:
- 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and reverse complements thereof.
An in vitro or an ex vivo method comprising:
- a) providing an isolated genetically engineered cell that comprises a gene editing multi-site (GEMS) sequence in said cell's genome, wherein said GEMS sequence comprises a plurality of nuclease recognition sequences, and wherein at least one of said plurality of nuclease recognition sequences is selected from the group consisting of sequences set forth in SEQ ID NOs:
- 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and reverse complements thereof;
- b) introducing into the isolated genetically engineered cell a nucleic acid vector comprising, in 5′ to 3′ order:
- i) a first donor flanking sequence homologous to a genomic sequence upstream of a selected nuclease recognition sequence from said plurality of nuclease recognition sequences in said GEMS sequence,
- ii) a donor nucleic acid sequence, and
- iii) a second donor flanking sequence homologous to a genomic sequence downstream of said selected nuclease recognition sequence;
- c) introducing into the isolated genetically engineered cell a guide polynucleotide;
- and
- d) introducing into the isolated genetically engineered cell a nuclease that recognizes said selected nuclease recognition sequence when bound to said guide polynucleotide.
An isolated gene editing multi-site (GEMS) construct that comprises a GEMS sequence, wherein said GEMS sequence comprises a sequence that is at least 80% identical to sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 84.
An isolated genetically engineered cell that comprises a gene editing multi-site (GEMS) sequence in said cell's genome, wherein said GEMS sequence comprises a sequence that is at least 80% identical to sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 84.
An in vitro or an ex vivo method comprising:
- a) providing an isolated genetically engineered cell that comprises a gene editing multi-site (GEMS) sequence in said cell's genome, wherein said GEMS sequence comprises a plurality of nuclease recognition sequences, and wherein said GEMS sequence comprises a sequence that is at least 80% identical to sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 84;
- b) introducing into the isolated genetically engineered cell a nucleic acid vector comprising, in 5′ to 3′ order:
- i) a first donor flanking sequence homologous to a genomic sequence upstream of a selected nuclease recognition sequence from said plurality of nuclease recognition sequences in said GEMS sequence,
- ii) a donor nucleic acid sequence, and
- iii) a second donor flanking sequence homologous to a genomic sequence downstream of said selected nuclease recognition sequence;
- c) introducing into the isolated genetically engineered cell a guide polynucleotide;
- and
- d) introducing into the isolated genetically engineered cell a nuclease that recognizes said selected nuclease recognition sequence when bound to said guide polynucleotide.
Dependencies are inferred from the claim text.