title: A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
abstract: Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.forename: Robert
authors.initials: R
authors.lastname: Morbitzer
authors.forename: Fabienne
authors.initials: F
authors.lastname: Lütge
authors.forename: Nadine
authors.initials: N
authors.lastname: Dannemann
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D005818:Genetic Engineering; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry; D000071336:Transcription Activator-Like Effectors
chemical_list: C434207:AvrBs4 protein, Xanthomonas campestris; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D000071336:Transcription Activator-Like Effectors; D003851:Deoxyribonucleases