Meganuclease-Based Artificial Transcription Factors

Abstract

Embedding middle-scale artificial gene networks in live mammalian cells is one of the most important future goals for cell engineering. However, the applications of the highly orthogonal and conventional artificial transcription factors currently available are limited. In this study, we present a scalable pipeline to produce artificial transcription factors based on homing endonucleases, also known as meganucleases. The introduction of mutations at critical sites for nuclease activity renders these homing endonucleases a simple but highly specific DNA binding domain for their specific DNA target. The introduction of inactivated meganucleases linked to transcriptional activator domains strongly induced reporter gene expression, while their fusion to transcriptional repressor domains suppressed them. In addition, we show that inactivated meganuclease-based transcription factors could be embedded in the synthetic membrane receptor synNotch and used to construct synthetic circuits. These results suggest that inactivated meganucleases are useful DNA-binding domains for the construction of synthetic transcription factors in mammalian cells.

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

“…Since the introduction of MNs application, unexpected drawbacks are constantly being discovered. Some of these challenges are: (i) The targeted locus must contain a specific MN cleavage site for each endonuclease whereas the microbial self-splicing intervening sequence could specifically duplicate into recipient alleles of their host gene lacking such sequence ( Rouet et al, 1994a ; Stoddard, 2011 ), (ii) low efficacy ( Chapdelaine et al, 2010 ), and (iii) potential genotoxicity ( Suzuki et al, 2020 ). The natural repertory of homing endonucleases is limited to a finite number of proteins, most of them still being hypothetical or uncharacterized.…”

Genome Editing: A Promising Approach for Achieving Abiotic Stress Tolerance in Plants

“…Meganucleases. Meganucleases are the endonucleases that can recognize and cut larger DNA sequences (>12 bp) in a sequence-specific manner [36,37]. They are reported to occur in a range of organisms including archaebacteria, bacteria, algae, fungi, yeast, and some of plant species.…”

“…Among these, the LAGLIDADG meganuclease (LMN) family has been extensively used for genome editing. Its name is derived from the sequence of the major motif present in the structure of this family of proteins [37,39]. LMNs are usually expressed in the chloroplast and mitochondria of unicellular eukaryotes.…”

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp

“…In early studies, regulation of synthetic gene circuits has mainly relied upon the use of natural TFs, such as TetR, LacI, and AraC . In recent years, progress has been made in the development of artificial orthogonal TFs that independently direct and regulate transcription of the targeted genes. ,− However, there is still a limited number of well-characterized orthogonal TFs . Therefore, the development of a new discipline of designing and building orthogonal TFs is urgently needed.…”