megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering

Abstract

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at ‘off-target’ sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate ‘megaTAL’, in which the DNA binding region of a transcription activator-like (TAL) effector is used to ‘address’ a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

“…Comparable frequencies of off-target mutagenesis were induced by the two types of meganuclease constructs, suggesting that the fused TAL effector had little impact on binding and cleavage activity of an engineered meganuclease at off-target sites, presumably because of high binding affinity of the stand-alone nuclease toward the noncognate DNA target sequences. These results are consistent with our earlier study of MegaTALs (21).…”

“…We next examined whether tethering engineered meganucleases to TAL effectors (as additional DNA binding domains) would improve the efficiency of targeted genome modification in human cells as recently described (21). The two redesigned meganucleases that targeted the cftr locus were each tethered to a TAL effector DNA binding domain (termed "NΔ148/C+63," a construct that spans from residue 149 of the canonical TAL effector N-terminal sequence to residue +63 beyond the end of the TAL effector repeat sequences).…”

“…Although an industrial-scale engineering pipeline for altering meganuclease specificity has been developed (6), the specialized knowledge and technology required for that approach generally precludes their use by academic laboratories. In addition, a recent study has demonstrated that MegaTALs (fusions of TAL effector DNA binding regions to meganucleases) can induce a high level of targeted gene modification in human cells (21). However, the utility of this platform still depends upon the development of efficient strategies to engineer meganucleases with desired specificity.…”

Genome-Editing Technologies: Principles and Applications

“…However, high-throughput TALE assembly methods have also been developed, including FLASH assembly (Reyon et al 2012), iterative capped assembly (Briggs et al 2012), and ligation independent cloning (Schmid-Burgk et al 2013), among others. More recent advances in TALEN assembly, though, have focused on the development of methods that can enhance their performance, including specificity profiling to uncover nonconventional RVDs that improve TALEN activity (Guilinger et al 2014a;Yang et al 2014;Juillerat et al 2015;Miller et al 2015), directed evolution as means to refine TALE specificity (Hubbard et al 2015), and even fusing TALE domains to homing endonuclease variants to generate chimeric nucleases with extended targeting specificity (discussed in more detail below) (Boissel et al 2014).…”

“…This overlap in form and function make their repurposing challenging, and limits their utility for more routine applications of genome editing. More recently megaTALsfusions of a rare-cleaving homing endonuclease to a TALE-binding domain-have been reported to induce highly specific gene modifications (Boissel et al 2014;Lin et al 2015a). These enzymes have enabled integration of antitumor and anti-HIV factors into the human CCR5 gene in both primary T cells and hematopoietic stem/progenitor cells (Sather et al 2015), as well as disruption of endogenous T-cell receptor elements in T cells (Osborn et al 2016), indicating their potential for enabling and enhancing immunotherapies.…”