A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

By Claudio Mussolino, Robert Morbitzer, Fabienne Lütge, N. Dannemann, T. Lahaye, T. Cathomen. Nucleic Acids Research, 2011

755 citations recorded by the source corpus.

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.

Subfields: Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/24cdc4e6bd20c51386e58311bb7eebfe612ceebb

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Citations in the corpus

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, background): “Moreover, the CCR5-specific TALEN was able to distinguish between the CCR5 target locus and a highly similar site in CCR2 when compared with ZFN technology.(37) By adopting TALEN gene editing technology, precise disruptions have also been introduced into the T cell receptor α constant (TRAC) gene an”
Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis (2014, background): “Off-targeting is a concern for both the ZFN (26) and TALEN (27) gene editing systems.”
Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice (2013, background): “knockout of the CCR5 gene for HIV resistance in human cells (21); disruption of the LDL receptor in swine (22); destruction of the bacterial blight disease susceptibility gene in rice (23); replacement of a tyrosine hydroxylase gene via TALEN-enhanced homologous recombination in zebrafish (24,25)].”
Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922 (2016, background): “In contrast, off-target effects were extremely rare in the event of one nucleotide mismatch for the TALENs-editing system [82, 83].”
TALENs: a widely applicable technology for targeted genome editing (2012, positive): “24 The TALEN framework was then refined by showing that nuclease activities could be greatly enhanced by truncating the length of this carboxy-terminal TALE-derived sequence 19 , 68 .”
Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases (2014, background): “and TALENs can induce mutations at off-target sites that are highly homologous to on-target sites (Gabriel et al. 2011; Mussolino et al. 2011; Pattanayak et al. 2011).”
Efficient TALEN-mediated gene knockout in livestock (2012, background): “In agreement with others, we found the truncated TALEN scaffold was superior for gene modification in primary fibroblasts (15, 20).”
TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction (2012, methodology): “In the case of TALENs, the optimum spacer length between the TALEN monomers depends on the architecture used (5,13,15,24).”