Profiling and Improving the Specificity of Site-Specific Nucleases

Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities.;Cas9 cleaves double-stranded DNA in cells at a sequence targeted by a short guide RNA (gRNA). We used in vitro selection to determine the abilities of eight Cas9:guide RNA complexes to cleave off-target DNA sequences. The selection results predicted five off-target substrates in the human genome that were confirmed to undergo genome cleavage in cells treated with Cas9:guide RNA complexes. To improve the specificity of Cas9:guided RNA complexes we describe the development of a FokI nuclease fusion to a catalytically dead Cas9 that requires simultaneous DNA binding and association of two FokI-dCas9 monomers to cleave DNA. Off-target DNA cleavage of the engineered FokI-dCas9 is further reduced by the requirement that only sites flanked by two gRNAs ~15 or 25 base pairs apart are cleaved. In human cells, fCas9 modified target and off-target DNA sites with comparable efficiency to wild-type Cas9 nucleases but with > 140-fold higher specificity.