MegaTALs

Meganucleases are enzymes in the endonuclease family that were discovered in the late 1980s. They are characterized by their capacity to recognize and cut large DNA sequences ranging from 14 to 40 base pairs, making them very specific. They are found more commonly in microbial species. MegaTALs typically have an 8–12 repeat TAL array appended to the N terminus, making them ~2.5 kb per monomer. Meganucleases can be used to target any plant or animal genome, although they have not been used extensively in many organisms including goats and sheep. MegaTALs have been able to achieve efficient gene editing with optimal reagents in cell culture lines where high-level delivery and toxicity are not a concern. Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14 bp), thus making them naturally very specific. The “MegaTev” is a recent architecture that has been generated and involves the fusion of the DNA-binding and cutting domain from a meganuclease (Mega, I-OnuI) with another nuclease domain derived from the GIY-YIG HEase (Tev, I-TevI). The megaTALs construct includes using a repeat variable diresidue (RVD) plasmid library and destination vector and protein linkers. MegaTALs combine the DNA-binding domains from TALENs with the high cleavage efficiency of meganucleases in a single chimeric protein. This creates large variants such as LHE, I-SceI, DmoCre, and E-Drel. MegaTALs can be used as a genome editing tool including the correction of variants that cause monogenic diseases, the enhancement of chimeric antigen receptor (CAR) T cell therapy, and cell-based regenerative medicine. Meganucleases have long recognition sequences, thus rendering them very specific, and this can be exploited to make site-specific DNA DSB in genome editing. However, meganucleases are insufficient to cover all possible target sequences. MegaTALs can be used as a genome editing tool, including the correction of variants that cause monogenic diseases. MegaTALs also have a therapeutic application in preventing entry of HIV into host CD4 cells through gene editing at the CCR5 locus of primary human T cells. MegaTALs have also been used for research on adoptive immunotherapy strategies, which are based on the retargeting of autologous T cells to recognize tumor antigens.