(116h) Engineering TAL Effector Nucleases (TALENs) for Targeted Genome Editing

Targeted genome editing enables researchers to disrupt, insert, or replace a genomic sequence precisely at a predetermined locus.  One well-established technology to edit a mammalian genome is known as gene targeting, which is based on the homologous recombination (HR) mechanism.  However, the low HR frequency in human cells prevents its wide application.  To address this limitation, a site-specific DNA double-strand break (DSB) can be introduced to the targeted genomic region, thus inducing the genome mortification efficiency by over 1000 fold. To generate a site-specific DSB in human genome, we need a DNA endonuclease that has two important properties: i) it must recognize a long DNA sequence with high specificity in order to avoid cytotoxic off-target DNA cleavage; ii) it must be readily designed to recognize and cleave a defined sequence in the genome. For that purpose, we constructed TAL effector nucleases (TALENs), which represent a new class of artificial DNA endonucleases.

TALENs utilize the central repeat units as the DNA recognition module and the FokI catalytic domain as the DNA cleavage module. Each central repeat unit consists of 34 amino acids, which are largely identical except for two highly variable amino acids at positions 12 and 13. Each repeat recognizes a single nucleotide, and the recognition specificity is determined by the two highly variable amino acids (e.g., NI recognizes A, HD recognizes C, HG recognizes T, and NN recognizes G or A). This simple DNA recognition code and its modular nature provide an ideal platform for constructing custom-designed artificial DNA nucleases. Although several TALEN scaffolds with different N- and C- terminal truncations have been tested by other groups, the optimum TALEN architecture that exhibits highest cleavage efficiency and bears minimal peptide portion has yet to be systematically determined. In this study, we carried out a systematic study testing ten various TALEN scaffolds against ten different DNA substrate configurations by the help of a yeast reporter system. According to the 10×10 matrix, we identified several TALEN scaffolds that are appropriate for therapeutic application.

As proof of concept, we constructed a pair of custom-designed TALENs to target a sequence within human β-globin (HBB) gene locus. A single nucleotide substitution (from A to T) in the codon for amino acid 6 on HBB gene leads to sickle cell disease, which is one of the most prevalent autosomal recessive disorders worldwide. This pair of TALEN with the optimized scaffolds allowed for efficient cleavage at the desired site in human cells and enhanced the gene targeting rate by >1000-fold with no detectable cytotoxicity. While all previous reported TALE recognition sites are preceded by a 5’-T at position 0, we discovered that certain TALEN variants are capable of recognizing and cleaving “unnatural” TALE binding sites preceded by A, C or G. The reduced 5’-nucleotide selectivity shown by our engineered TALEN variants allows for greater flexibility in choosing desired target site. Above all, due to the ease in design and construction, the high DNA cleavage activity, and the low cytotoxicity, the engineered TALENs represent a powerful tool for targeted genome editing.