THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

Description

RELATED APPLICATIONS[0001]This application is a continuation-in-part of PCT Application No. PCT / US2014 / 033082, filed Apr. 4, 2014, which claims the benefit of U.S. Provisional Application Ser. No. 61 / 808,594, filed Apr. 4, 2013, the teachings of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT[0002]This invention was made with government support under R01-HL118744, R00-HL098364 and R01-DK095384 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9...

AI Technical Summary

Benefits of technology

[0004]Work described herein demonstrates methods of allele targeting using CRISPR / Cas systems resulting in mutant cells with efficiencies of up to 80%. In particular, work described herein surprisingly and unexpectedly demonstrates that a multiple guide strategy (e.g., using two or more ribonucleic acids which guide Cas protein to and hybridize to a target polynucleotide sequence) efficiently and effectively deletes target polynucleotide sequences (e.g., B2M, HPRT, CCR5 and / or CXCR4) in primary somatic cells (e.g., human blood cells, e.g., CD34+ and T cells), in contrast to a single guide strategy which has been demonstrated by the inventors to efficiently delete target polynucleotide sequences in cell lines (e.g., 293T) but not in primary somatic cells. These vastly improved methods permit CRISPR / Cas systems to be utilized effectively for the first time for therapeutic purposes. Methods of delivery of CRISPR / Cas systems to human stem cells are provided. In addition, methods of specifically identifying useful RNA guide sequences are provided, along with particular guide sequences useful in targeting specific genes (e.g., B2M, HPRT, CCR5 and / or CXCR4). Moreover, methods of treatment (e.g., methods of treating HIV infection) utilizing the compositions and methods disclosed herein are provided.

[0005]In some aspects, the present invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

Problems solved by technology

Thus, CRISPR / Cas systems could be useful tools for therapeutic applications, but unfortunately prior published reports have demonstrated an efficiency of allele targeting of only 2%-4% in human stem cells (Mali et al., Science 339:823-826 (2013)).

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