The General Hospital Corporation

St. Jude Children's Research Hospital

Shengdar Q Tsai

J Keith Joung

James Angstman

Benjamin Kleinstiver

Morgan Maeder

Jeffry D Sander

Jason Michael Gehrke

Rebecca Tayler Cottman

Cicera R Lazzarotto

Keith J Joung

The present invention relates to methods to improve the absolute rate of homology-directed repair (HDR) and/or to improve the relative rate of HDR compared with non-homologous end joining (NHEJ).

Methods for increasing efficiency of nuclease-induced homology-directed repair

Unbiased, genomewide and highly sensitive methods for detecting mutations, e.g., off-target mutations, induced by engineered nucleases.

Genomewide unbiased identification of DSBs evaluated by sequencing (GUIDE-Seq)

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems.

Increasing specificity for RNA-guided genome editing

The invention relates to a high-throughput method for characterizing the genome-wide activity of editing nucleases in vitro.

High-throughput method for characterizing the genome-wide activity of editing nucleases in vitro (Change-Seq)

Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided FokI Nucleases (RFNs), e.g., FokI-Cas9 or FokI-dCas9-based fusion proteins.

Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing

Sensitive, unbiased methods for genome-wide detection of potential off-target nuclease cleavage sites in DNA, e.g., in cell type-specific genomic DNA samples.

Full interrogation of nuclease DSBs and sequencing (FIND-seq)

High-throughput method for characterizing the genome-wide activity of editing nucleases in vitro

Sensitive, unbiased methods for genome-wide detection of potential CRISPR-Cas9 off-target cleavage sites from cell type-specific genomic DNA samples.

Comprehensive in vitro reporting of cleavage events by sequencing (CIRCLE-seq)

Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided Foki Nucleases (RFNs), e.g., Fokl-Cas9 or Foki-dCas9-based fusion proteins.

Methods and constructs for the multiplex expression of highly active CRISPR guide RNAs (gRNAs) from RNA Polymerase II and III promoters, optionally in mammalian cells. The present invention is based, at least in part, on the discovery that Csy4, an endoribonuclease that recognizes a short RNA hairpin sequence, can be used to cleave out multiple functional gRNAs encoded on a single longer RNA transcript (produced from an RNA pol II or III promoter) in which the individual gRNAs are separated by Csy4 cleavage sites.

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Memphis, TN, United States of America

Shengdar Q Tsai