Optimized gene editing utilizing a recombinant endonuclease system

Patent metadata

Field	Value
Publication number	US-11535871-B2
Application number	US-201615573732-A
Country	US
Kind code	B2
Filing date	May 13, 2016
Priority date	May 14, 2015
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

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.

First claim

The invention claimed is:

1. 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.

2. The method of claim 1 , wherein the donor DNA comprises the sequence of SEQ ID NO:3 and the one or more sgRNAs comprises the sequence of SEQ ID NO:6,
the donor DNA comprises the sequence of SEQ ID NO:4 and the one or more sgRNAs comprises the sequence of SEQ ID NO:7, or
the donor DNA comprises the sequence of SEQ ID NO:5 and the one or more sgRNAs comprises the sequence of SEQ ID NO:8.

3. The method of claim 1 , wherein the fusion protein further comprises a nuclear localization signal (NLS) sequence.

4. The method of claim 1 , wherein the fusion protein further comprises a fluorescent labeled protein.

5. The method of genomic editing of claim 1 , wherein the donor DNA further comprises an expression cassette and two flanking sequences, and wherein the sequence configured for binding the zinc finger domain is not within the expression cassette.

6. The method of claim 5 , wherein the sequence configured for binding the zinc finger domain is positioned between the two flanking sequences in the donor DNA.

7. The method of claim 6 , wherein the two flanking sequences are each at least 10 base pairs in length, and homologous to sequences in the genome of the population of cells.

8. The method of claim 1 , wherein contacting the population of cells comprises performing a technique selected from the group consisting of: transfection, electroporation, and transformation.

9. The method of claim 1 , wherein the population of cells comprise stem cells or progenitor cells.

10. The method of claim 7 , wherein the two flanking sequences are each at least 80 base pairs in length, wherein a first of the two flanking sequences is positioned 5′ and a second of the two flanking sequences is positioned 3′ within the donor DNA.

11. The method of claim 1 , wherein (i) the donor DNA, (ii) the one or more sgRNAs or the vector encoding the one or more sgRNAs, or (iii) both (i) and (ii), is premixed with the one or more vectors encoding the fusion protein before contacting the population of cells.

Assignees

• Univ Southern California

Inventors

• Junge Jason
• Hunt Timothy
• Fraser Scott E

Classifications

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C07K2319/81

  containing a Zn-finger domain for DNA binding · CPC title

• C07K14/43504

  from invertebrates · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title