Efficient non-meiotic allele introgression
Inventors
Fahrenkrug, Scott C. • CARLSON, DANIEL F.
Assignees
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Publication Number
US-9528124-B2
Publication Date
2016-12-27
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 invention relates to a method of introgressing an allele or gene into chromosomal DNA of a non-human animal cell. The method introduces into an isolated non-human animal cell a CRISPR/Cas endonuclease, a guide RNA (gRNA) with a spacer sequence that interacts with a target sequence in the chromosomal DNA, and a homology-directed repair (HDR) template DNA sequence encoding the allele or gene flanked by sequences homologous to the target sequence in the cell’s chromosomal DNA.
A key refinement is that the HDR template DNA 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. The mismatch is introduced into the chromosomal DNA of the cell so that the resulting chromosomal DNA sequence is not found in the non-human animal line.
The disclosed approach further emphasizes efficient non-meiotic allele introgression into livestock cells using targeted endonucleases with HDR templates, including precise SNP and small-indel edits. The disclosed work includes supporting examples across pig, goat, and cattle cell contexts, including allele introgression into fibroblasts and generating biomedical model pigs with DAZL and APC edits, and describes an HDR landing-pad based workflow for transgene insertion that uses a TALEN-HDR/RMCE workflow.
Claims Coverage
The independent claim (clm-00001) is directed to a method for introgressing an allele or gene into chromosomal DNA of a non-human animal cell using CRISPR/Cas endonuclease, a gRNA, and an HDR template that encodes a mismatch altering gRNA interaction, where the mismatch results in a chromosomal sequence not found in the non-human animal line. Across dependent claims identified in the partial content, the main inventive features are further refined by specifying mismatch types and constraints, specifying allele-source relationships and trait categories for introgression outcomes.
CRISPR/Cas HDR template flanked by homology for allele introgression
Introducing a CRISPR/Cas endonuclease, a guide RNA (gRNA) comprising a spacer RNA sequence that interacts with a target sequence in the chromosomal DNA, and an HDR template DNA sequence encoding an allele or gene flanked by sequences homologous to the target sequence, such that the chromosomal DNA alters to have identity with the HDR template DNA sequence at the target sequence, thereby introgressing the allele or gene into the chromosomal DNA.
HDR-encoded mismatch that alters gRNA spacer interaction and yields a sequence not found in the animal line
The HDR template DNA sequence also 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 cell that is not found in the non-human animal line.
Mismatch as a base substitution that alters gRNA binding behavior
The mismatch is created by substituting a DNA base for a base that does not promote binding to the gRNA.
Mismatch size constraint as a 1 to 5 base-pair substitution
The mismatch includes a substitution consisting of a 1 to 5 base-pair substitution.
Mismatch characterized by insertion or deletion of a DNA base
The mismatch involves an insertion or a deletion of a DNA base.
Mismatch specified as an SNP located within the homologous allele region
Using the HDR template to perform homologous replacement of an endogenous allele in a cell with a homologous allele, where the mismatch is a single nucleotide polymorphism (SNP) located within the allele that is homologous to the endogenous allele.
Trait-linked allele replacement for horn growth, meat/milk/dairy, or disease resistance
Replacing an endogenous allele in a cell with a homologous different allele via the HDR template to alter a trait, where the trait is selected from horn growth, meat production, milk production, dairy, or disease resistance traits.
The claim set is centered on CRISPR/Cas-mediated HDR introgression using an HDR template that encodes a mismatch to alter gRNA interaction, producing an edited chromosomal sequence not present in the non-human animal line. Dependent claims further define mismatch form (substitution vs insertion/deletion), mismatch scale (1 to 5 base pairs), mismatch identity (SNP), mismatch location (within the homologous allele region), and trait categories tied to the allele replacement outcomes.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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