Efficient non-meiotic allele introgression
Inventors
Fahrenkrug, Scott C. • CARLSON, DANIEL F.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10959414-B2
Publication Date
2021-03-30
Expiration Date
Abstract
Methods, uses, and animals for introgression of alleles between animals, including SNPs. One embodiment involves introducing a targeted targeting endonuclease system and a HDR template into a cell with a mismatch in the binding of the targeting endonuclease and the targeted site.
Core Innovation
The disclosure describes efficient non-meiotic allele introgression in which a targeting endonuclease system, including CRISPR/Cas9 endonucleases and TALEN in described examples, cleaves chromosomal DNA at a target sequence in an isolated cell. A homology-directed repair template sequence is provided with sequences homologous to the target sequence so that an allele or a gene is inserted into the cleavage site and introgressed into the chromosomal DNA of the cell, supporting the creation of live animals.
A key feature is that the HDR template comprises an intentional DNA mismatch in the target sequence that alters the interaction with the RNA spacer sequence of the gRNA. The mismatch is introduced into the chromosomal DNA of the cell, creating a sequence in the animal that is not found in the non-human animal line, with the resulting animal having a phenotypic difference relative to the non-human animal line.
The disclosure further describes live animal generation based on cells carrying the introgressed genetic changes, including marker-free and reporter-free or selection-marker-free approaches. It also describes related concepts such as engineered mismatches intended to stabilize SNP introgression, landing-pad/RMCE concepts, and examples across livestock and other species, with emphasis on insertion of small edits such as SNPs and small indels and maintaining edited outcomes over extended culture.
Claims Coverage
The partial content provides one independent claim: a method of making a non-human animal with genome introgression using CRISPR/Cas9-mediated HDR in an isolated cell followed by embryo production and implantation. The inventive scheme is further narrowed in dependent claims by specifying mismatch type and size and linking the edited locus or allele to traits, and by adding breed-related constraints for the replacement allele.
CRISPR/Cas9 HDR-mediated introgression into an isolated cell
Introducing into a cell isolated from a non-human animal line a CRISPR/Cas9 endonuclease and a guide RNA with a spacer RNA sequence that interacts with a target sequence in chromosomal DNA, together with an HDR template flanked by sequences homologous to the target sequence so that the HDR template is inserted into the cleavage site to cause the chromosomal DNA of the cell to have identity with the HDR template sequence at the target sequence, thereby introgressing the allele or the gene into the chromosomal DNA of the cell.
Introgression using an HDR template mismatch altering gRNA spacer interaction
Wherein the HDR template sequence comprises a DNA sequence encoding a mismatch in the target sequence that alters interaction with the RNA spacer sequence of the gRNA, wherein the mismatch is introduced into the chromosomal DNA of the cell and creates a sequence in the chromosomal DNA of the animal that is not found in the non-human animal line, and wherein the non-human animal has a phenotypic difference relative to the non-human animal line.
Embryo production and surrogate implantation of the edited cell
Introducing the cell resulting from the CRISPR/Cas9 and HDR steps into an enucleated oocyte of the same species as the non-human animal line to produce an embryo, and implanting the embryo in a surrogate mother to produce the non-human animal.
Mismatch size/type and trait-linked allele introgression
Dependent claim refinements include that the mismatch comprises a 1 to 5 base pair substitution, that the mismatch comprises an insertion or a deletion of a DNA base, and that the mismatch is a single nucleotide polymorphism located within the homologous allele region. Additional dependent refinements connect the target sequence and replacement allele to loci associated with traits, including horn growth, meat or meat production, milk or milk production, dairy, or disease resistance.
Species line and replacement allele breed constraint
Dependent claim refinements specify that the non-human animal line is selected from swine, cattle, sheep, non-human primate, mouse, and rat lines, and include a constraint that the homologous replacement allele replaces an endogenous allele with a replacement allele not from the same breed of animal as the non-human animal line.
Overall, the claim coverage centers on producing a non-human animal in which an allele or gene is introgressed into chromosomal DNA via CRISPR/Cas9-mediated HDR using an HDR template that intentionally encodes a mismatch altering gRNA spacer interaction, leading to a sequence not found in the original line and a phenotypic difference. The dependent claims refine mismatch content and size and relate edited loci to trait categories and lineage or breed-related constraints.
Stated Advantages
Efficient non-meiotic allele introgression is described.
The disclosure emphasizes precise, high-frequency small edits such as SNP or small indel editing and stabilization of intended SNP introgression.
Marker-free and reporter-free and selection-marker-free genome editing approaches are described.
Edited outcomes are described as stable over extended culture.
The approach is described as enabling generation of live animals from edited loci in mammalian models, including livestock and other species.
Documented Applications
Non-meiotic introgression of a POLLED allele into horned fibroblasts and generation of hornless dairy cattle.
Introgression of specific trait-associated loci in livestock models, including GDF8 (G938A) and p65 S531P (XmaI RFLP), and LDLR examples.
Generation of live animals from edited porcine loci, including DAZL and APC models.
Engineered mismatch strategies are described for introgressing SNPs and small edits in species including swine, goat, and cattle.
Landing-pad and RMCE concepts are described in the context of the disclosed editing and live animal generation.
Interested in licensing this patent?