| Summary: | Designer effectors based on the DNA binding domain (DBD) of <i>Xanthomonas</i> transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17 <i>Xanthomonas</i> TALE repeats with TALE-like units from the bacterium <i>Burkholderia rhizoxinica</i>. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the <i>CCR5</i> and <i>CXCR4</i> loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized <i>CXCR4</i>-specific epigenome editor resulted in rapid silencing of endogenous <i>CXCR4</i> expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome.
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