Targeted non-viral DNA insertions

Abstract

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

Description

PRIOR RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 520,117 filed on Jun. 15, 2017 and U.S. Provisional Application No. 62 / 552,180 filed on Aug. 30, 2017, both of which are hereby incorporated by reference in their entireties.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT[0002]This invention was made with government support under grant no. P50 GM082250 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]The ability to introduce small mutations (indels) at targeted sites in the genome of cells by electroporating a Cas9-gRNA complex (RNP) into the cells has been developed. However, since these mutations are random and introduced by non-homologous end joining, they can cause a protein to be knocked out of frame (Schumann et al. PNAS 112(33): 10437-10442 (2015)). Other methods have been developed to introduce a...

AI Technical Summary

Benefits of technology

[0004]The present invention is directed to compositions and methods for editing the genome of a cell. The inventors have discovered that large nucleotide sequences, for example, sequences greater than about 200 nucleotides in length, can be inserted into a targeted region in the genome of a cell. In some methods, integration of sequences greater than about 200 nucleotides in length occurs while reducing off-target effects and / or reducing loss of cell viability.

Problems solved by technology

However, since these mutations are random and introduced by non-homologous end joining, they can cause a protein to be knocked out of frame (Schumann et al.

However, since the size of these oligonucleotides is limited to the length of DNA that can be chemically synthesized (

In addition to size limitations, it is well established that electroporation of naked DNA, in particular, naked DNA larger than about 200 bps, into cells often leads to massive cell death owing to the activation of intrinsic cellular defense mechanism (Cornu et al.

Although non-integrating viral vectors, such as integrase defective lentiviral vectors or adeno-associated viral (AAV) vectors, have been used to deliver large donor nucleic acid sequences to cells, these vectors require viral infection and cause off-target effects.

Images