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AssigneeThe Regents of the University of California
InventorsEthan Bier, Annabel Guichard, Valentino Gantz
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An active genetic approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. Embodiments are provided for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located at some distance from the gRNA cut site. A lethal mosaicism is provided that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process provide a shadow-drive, that are applicable to improvements in health and agriculture.
A method of introducing a preferred nucleotide sequence into a genome by allelic-drive copy-cutting, comprising genomically replacing a non-preferred nucleotide sequence with that of a preferred allele of the same gene using an allelic-drive element that comprises first and second guide RNAs and a cleavage resistant preferred allele, wherein a Cas endonuclease guided by the first guide RNA copies the allelic-drive element, and a Cas endonuclease guided by the second guide RNA cuts the non-preferred allele, but not the preferred allele, and copies the cleavage resistant preferred allele into a double stranded DNA break created by the second guide RNA by homology-directed repair (HDR)-mediated repair.
A method of introducing a preferred nucleotide sequence into a genome by allelic-drive copy-grafting, comprising genomically replacing a non-preferred nucleotide sequence with that of a preferred allele of the same gene using an allelic-drive element that comprises a first and a second guide RNAs and a cleavage resistant site adjacent to the preferred allele, wherein Cas endonuclease guided by the first guide RNA copies the allelic-drive element, and the second guide RNA cuts the non-preferred allele, but not the preferred allele, that is associated with the cleavage resistant sequence residing within less than 100 nucleotides of the preferred allele, and copies the cleavage resistant sequence together with the preferred adjacent allele by virtue of a short range single stranded resection step which occurs during homology-directed repair (HDR)-mediated repair.
A method of selectively eliminating an allele of a gene generated by non-homologous end joining (NHEJ) comprising utilizing a lethal mosaicism technique, wherein NHEJ-induced drive-resistant, non-functional, or loss-of-function alleles of an essential gene are dominantly eliminated in progeny due to maternal perdurance of Cas/guide RNA complexes targeting the paternal allele.
A method of germline editing comprising repair of a cleavage-sensitive allele with sequences provided by a cut-resistant allele present on the homologous chromosome in heterozygous individuals.
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