US 11,535,871 B2Granted

Optimized gene editing utilizing a recombinant endonuclease system

AssigneeUniversity of Southern California

InventorsJason Junge, Timothy Hunt, Scott E. Fraser

Life of this patent

Estimating the term…

Loading patent…

Abstract

Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.

Claims11

Dependent claims

A method of genomic editing comprising:

contacting a population of cells with a quantity of one or more vectors encoding a fusion protein, said fusion protein comprising a clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) 9 and a zinc finger domain, wherein the one or more vectors encoding the fusion protein comprises the sequence of SEQ ID NO:1 or encodes the fusion protein of the sequence of SEQ ID NO:2,
contacting the population of cells with one or more single guide RNAs (sgRNAs) or a vector encoding the one or more sgRNAs, and
contacting the population of cells with a donor DNA, said donor DNA comprising a sequence configured for binding the zinc finger domain,
wherein the Cas9 of the fusion protein induces a double stranded break (DSB), which permits homologous recombination (HR) and/or non-homologous end joining (NHEJ) of the DSB, and the donor DNA is brought in proximity to the site of the DSB for integration and thereby editing the genome of the population of cells.

1 more element

Dependencies are inferred from the claim text.

Facts

Application: 15/573,732Filed 13 May 2016 · us provisional application
Priority: 14 May 2015
Published: US 2018/0127785 A110 May 2018
Granted: 27 Dec 2022
Term ends: Not estimatedThe source records don’t support an estimate.

Classification14 codes

C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12N 15/907
Using homologous recombination: in mammalian cells
C07K 14/43504
From animals; from humans: from invertebrates
C12N 9/222
Ribonucleases [RNase]; Deoxyribonucleases [DNase]: clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes