TAR Cloning: Perspectives for Functional Genomics, Biomedicine, and Biotechnology

Abstract

Completion of the human genome sequence and recent advances in engineering technologies have enabled an unprecedented level of understanding of DNA variations and their contribution to human diseases and cellular functions. However, in some cases, long-read sequencing technologies do not allow determination of the genomic region carrying a specific mutation (e.g., a mutation located in large segmental duplications). Transformation-associated recombination (TAR) cloning allows selective, most accurate, efficient, and rapid isolation of a given genomic fragment or a full-length gene from simple and complex genomes. Moreover, this method is the only way to simultaneously isolate the same genomic region from multiple individuals. As such, TAR technology is currently in a leading position to create a library of the individual genes that comprise the human genome and physically characterize the sites of chromosomal alterations (copy number variations [CNVs], inversions, translocations) in the human population, associated with the predisposition to different diseases, including cancer. It is our belief that such a library and analysis of the human genome will be of great importance to the growing field of gene therapy, new drug design methods, and genomic research. In this review, we detail the motivation for TAR cloning for human genome studies, biotechnology, and biomedicine, discuss the recent progress of some TAR-based projects, and describe how TAR technology in combination with HAC (human artificial chromosome)-based and CRISPR-based technologies may contribute in the future.

Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli

“…The new BAC vectors, together with the modernized cloning methods demonstrated in this study, will accelerate innovation in the field of genome synthetic biology. Our technologies would be combined with other methods including CRISPR-Cas-based systems, transformation-associated recombination (TAR) cloning in yeast, , refined human artificial chromosome (HAC) vectors, , rapid giant DNA production in Bacillus subtilis , multiplex genome editing by natural transformation (MuGENT) in Vibrio species, α-proteobacterial chassis , and Streptomyces chassis, and a cell-free cloning system (OriCiro Genomics, Inc.) Importantly, megabase-sized synthetic chromosomes assembled in E. coli can be directly transferred via conjugation machinery.…”

Human Artificial Chromosomes and Their Transfer to Target Cells

“…Next, the multimerized alpha-satellite DNA repeats and linearized vector for TAR cloning were transferred to Saccharomyces cerevisiae yeast cells, where the DNA repeats recombined with each other. This event resulted in the formation of longer sequences that were inserted into the TAR vector containing the blasticidin resistance gene [ 47 , 48 , 49 , 50 ]. The resulting constructs were transferred to HT1080 human fibrosarcoma cells, where they additionally multimerized and formed circular DNA molecules 1–2.5 Mbp in length.…”

Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters

“…Using pCAP05, the violacein BGC (∼8 kb) from marine bacterium Pseudoalteromonas luteoviolacea was cloned and expressed in Pseudomonas putida and Agrobacterium tumefaciens ( Zhang J. J. et al, 2017 ). Overall, TAR has been widely employed for BGC cloning, leading to the identification of many novel NPs ( Alberti et al, 2019 ; Kouprina and Larionov, 2019 ; Table 2 ).…”