Manuel Rhiel, Kerstin Geiger, Geoffroy Andrieux, Julia Rositzka, Melanie Boerries, Toni Cathomen and Tatjana I. Cornu

Frontiers in Genome Editing; PUBLISHED 20 February 2023; DOI 0.3389/fgeed.2023.1130736

ABSTRACT

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.

See https://www.frontiersin.org/articles/10.3389/fgeed.2023.1130736/full

FIGURE 1: CAST-Seq analysis for CCR5-targeting TALEN. (A) NGS-based genotyping of CCR5 and CCR2. Indicated is the fraction of alleles bearing Indels at the CCR5 on-target site and the off-target site in CCR2 in untreated (UT) T cells, and T cells edited with TALEN. (B) Structural variations. Circos plot illustrates CAST-Seq results with enlargement of the chromosome three region encompassing CCR5 and CCR2 loci. Lines represent chromosomal rearrangements with the CCR5 target site: OMTs with >20 hits in red, NBSs with >40 hits in grey, ambiguous classification (OMT/HMT) in yellow. Red and blue layers represent the alignment and homology scores, respectively. (C) Alignment. Illustrated is the CAST-Seq alignment for ANKRD55 (OMT#1) to two right TALEN arms. Mismatched bases are highlighted in red. Positions of primers P#1/P#2 for NGS validation are indicated. (D) Genotyping of ANKRD55. NGS was performed with primers P#1/P#2. (E) Coverage plot of ANKRD55. Plot shows chromosomal position vs. number of reads, the putative TALEN binding sites (green and purple dashed lines), as well as the positions of primers P#1/P#2 and P#3/P#4. Red vertical lines show boundary of region. (F) Genotyping of OMT#1. NGS was performed with primers P#3/P#4. *** specifies p-value<0.001 (Fisher’s exact test, Bonferroni corrected p-values).