Efficient non-meiotic allele introgression in livestock
Inventors
Fahrenkrug, Scott C. • CARLSON, DANIEL F.
Assignees
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Publication Number
US-11477969-B2
Publication Date
2022-10-25
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 disclosed invention provides a method for making a non-human animal in which an allele or a gene is introgressed into the chromosomal DNA of a cell by homology-directed repair (HDR) following cleavage by CRISPR/Cas9 at a target sequence. A CRISPR/Cas9 endonuclease together with a guide RNA (gRNA) comprising a spacer RNA sequence that interacts with the target sequence is introduced into a cell isolated from a non-human animal line, and an HDR template sequence encoding the allele or gene flanked by sequences homologous to the target sequence is introduced.
The HDR template sequence comprises a DNA sequence encoding a 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 chromosomal DNA of the animal that is not found in the non-human animal line.
The resulting non-human animal has a phenotypic difference relative to the non-human animal line. The method further includes 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, followed by implanting the embryo in a surrogate mother to produce the non-human animal.
Claims Coverage
The document contains one independent claim, with dependent claims refining mismatch and allele/recombination parameters. The independent claim centers on introgressing an allele or gene via CRISPR/Cas9-mediated HDR using an HDR template that includes a mismatch to alter gRNA interaction, yielding a phenotypic difference and a sequence not found in the starting line.
CRISPR/Cas9 cleavage and HDR template insertion for allele/gene introgression
Introducing into a cell isolated from a non-human animal line a CRISPR/Cas9 endonuclease, a guide RNA comprising a spacer RNA sequence that interacts with a target sequence in chromosomal DNA, and a homology-directed repair (HDR) template sequence encoding an allele or a gene flanked by sequences homologous to the target sequence, wherein cleavage occurs at the target sequence and the HDR template sequence is inserted into the cleavage site so the chromosomal DNA has identity with the HDR template sequence at the target sequence.
Mismatch in HDR template alters gRNA interaction and creates a sequence not found in the starting line
The HDR template sequence also comprises a DNA sequence encoding a mismatch in the target sequence that alters the 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.
Phenotypic difference and non-human animal production via enucleated oocyte and surrogate implantation
The mismatch and HDR introgression produce a non-human animal having a phenotypic difference relative to the non-human animal line, wherein the method includes introducing the resulting cell into an enucleated oocyte of the same species to produce an embryo and implanting the embryo in a surrogate mother to produce the non-human animal.
Applicable non-human animal line selection
The non-human animal line is selected from a swine line, a cattle line, a sheep line, a non-human primate line, a mouse line, or a rat line.
Overall, the claims cover making a non-human animal by CRISPR/Cas9 cleavage and HDR-mediated insertion of an allele or gene using an HDR template that contains a mismatch altering gRNA interaction, thereby creating a sequence not found in the starting line and resulting in a phenotypic difference after enucleated oocyte embryo production and surrogate implantation.
Stated Advantages
Creates a sequence in the chromosomal DNA of the animal that is not found in the non-human animal line.
Produces a non-human animal with a phenotypic difference relative to the non-human animal line.
Documented Applications
Making a non-human animal using CRISPR/Cas9 and HDR in specified non-human animal lines, followed by embryo production in an enucleated oocyte and surrogate implantation.
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