Chimerization Enables Gene Synthesis and Lentiviral Delivery of Customizable TALE-Based Effectors.

Yongxing Fang,Wladislaw Stroukov,Claudio Mussolino,Toni Cathomen

doi:10.3390/ijms21030795

Abstract

Designer effectors based on the DNA binding domain (DBD) of Xanthomonas 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 Xanthomonas TALE repeats with TALE-like units from the bacterium Burkholderia rhizoxinica. 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 CCR5 and CXCR4 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 CXCR4-specific epigenome editor resulted in rapid silencing of endogenous CXCR4 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.

Highlights

The availability of platforms capable of binding to predefined sites in the human genome enables the targeted activity of designer effectors at predefined sites
We reasoned that minimizing the repetitiveness of the DNA sequence coding for a transcription activator-like effectors (TALEs)-DNA binding domain (DBD) would allow gene synthesis and lentiviral delivery of the corresponding effectors
Sequence diversification was achieved by (i) optimization of the codon usage to maximize expression in mammalian systems using a tool available online (Integrated DNA technology, Integrated DNA Technology (IDT)), (ii) introducing amino acid changes at positions 4, 11, 24, and 32, which are polymorphic in natural TALE-DBD of Xanthomonas bacteria [8], and (iii) manual optimization of the remaining repetitive DNA sequences that are longer than eight base pairs where possible

Summary

Introduction

The availability of platforms capable of binding to predefined sites in the human genome enables the targeted activity of designer effectors at predefined sites. The DNA binding domains derived from transcription activator-like effectors (TALE) of Xanthomonas bacteria have been extensively used, in the last decade, for their high specificity and simplicity for generating novel DBDs [6], and to date, representing one of the most successful tools to target specific sequences in the large mammalian genome. The strength of this DNA binding domain lies in its structure, i.e., a variable length array of 15.5 to 17.5 modules each one capable of binding to a single nucleotide of the target sequence. This in turn has prevented the widespread use of this technology far

Methods

Results

Conclusion