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Publication Number
US-12070022-B2
Publication Date
2024-08-27
Expiration Date
Abstract
The present disclosure relates to methods for making genetic edits in vitro in a non-human vertebrate cell or embryo at a plurality of target chromosomal DNA sites. Methods for making a non-human animal having multiplex genetic edits at a plurality of target chromosomal DNA sites and making a non-human vertebrate animal chimeric for host cells and donor cells are also considered.
Core Innovation
The disclosed invention provides a method of making gene edits in vitro in a non-human vertebrate cell at two or more gene loci. The method includes simultaneously introducing a first targeted CRISPR endonuclease system directed to a first target chromosomal DNA site together with a first homology directed repair template homologous to a single gene locus at the first target site, and a second targeted CRISPR endonuclease system directed to a second target chromosomal DNA site together with a second homology directed repair template homologous to a single gene locus at the second target site.
In the method, the first homology directed repair template sequence independently replaces a native chromosomal DNA sequence at the first gene locus, and the second homology directed repair template sequence independently replaces a native chromosomal DNA sequence at the second gene locus. By performing these independent replacements at two or more loci, the method makes a non-human vertebrate cell comprising gene edits at two or more gene loci.
The disclosure addresses the need to generate multiple precise gene edits at multiple loci in non-human vertebrate cells and embryos, including obtaining multiple homology-directed repair events at distinct gene loci in vitro. It emphasizes multiplexed homology directed repair edits at multiple loci using simultaneous targeted endonucleases and corresponding HDR templates, including knockouts and allele replacement outcomes.
The document also describes using multiplex gene editing to create non-human vertebrate animals with multiple knockouts and/or sterility through multiplex disruption of genes related to gametogenesis and spermatogenesis. It additionally describes producing chimeric animals via host niche complementation models, and switching inheritance so donor cell genetics are passed via nonfunctional gametes in the host.
Claims Coverage
The partial content includes one independent claim covering simultaneous in vitro multiplex gene editing at two or more gene loci in a non-human vertebrate cell using two targeted CRISPR endonuclease systems and corresponding homology directed repair templates. The independent claim comprises two inventive features focused on independent replacement at separate gene loci and the resulting multiplexed edited cell.
Simultaneous multiplex CRISPR endonuclease with independent HDR template replacements
Introducing simultaneously into a non-human vertebrate cell a first targeted CRISPR endonuclease system directed to a first target chromosomal DNA site and a first homology directed repair template homologous to a single gene locus at the first site, and a second targeted CRISPR endonuclease system directed to a second target chromosomal DNA site and a second homology directed repair template homologous to a single gene locus at the second site, wherein each HDR template sequence independently replaces a native chromosomal DNA sequence at its respective gene locus.
Gene edits at two or more gene loci in a non-human vertebrate cell
Making a non-human vertebrate cell comprising gene edits at two or more gene loci based on the simultaneous introducing step of the first and second CRISPR endonuclease systems and their corresponding homology directed repair templates.
Overall, the independent claim requires simultaneous introduction of two targeted CRISPR endonuclease systems into a non-human vertebrate cell along with corresponding HDR templates that independently replace native sequences at distinct single gene loci, thereby producing a cell with gene edits at two or more loci.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Creating F0 founder animals with multiple knockouts.
Producing chimeric animals via host niche complementation, including failure-to-thrive and immunodeficiency models.
Switching inheritance so donor cell genetics are passed via nonfunctional gametes in the host.
Inducing sterility via multiplex disruption of gametogenesis and spermatogenesis genes.
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