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

Natalay Kouprina,Vladimir Larionov

doi:10.1016/j.omtm.2019.05.006

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.

Highlights

The ability to quickly and accurately isolate full-length genes, including coding as well as non-coding regions that participate in controlling gene expression, has become especially relevant in recent years in light of the growing interest in gene function studies and gene therapy
Application of the transformation-associated recombination (TAR) cloning strategy has been demonstrated for long-range haplotyping,[33,34,35,36,37] and, as a result, this technique is well suited for large-scale analysis of haplotypes in multiple heterozygous individuals to identify haplotypes that might contribute to disease
Accurate long-read sequencing of 13 TAR isolates of rDNA units covers 0.82 Mb of the chromosome 21 rDNA cluster.[42] dDirect isolation of a set of overlapping 50- to 120-kb genomic segments by in vivo recombination in yeast was used to perform a mutational analysis of the 750-kb region in X-linked families

Summary

16 Molecular Therapy

Great apes, the mouse, and other organisms (Table 1).[11,12,13,14,15,18,19,20,21,22,23,29,30,31,32]. Accurate long-read sequencing of 13 TAR isolates of rDNA units covers 0.82 Mb of the chromosome 21 rDNA cluster.[42] dDirect isolation of a set of overlapping 50- to 120-kb genomic segments by in vivo recombination in yeast was used to perform a mutational analysis of the 750-kb region in X-linked families These results excluded the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy.[36] the last decade because of advances in genome editing.[48] development of the CRISPR system created the opportunity to site- correct mutated DNA base pairs or insert a corrective cDNA minigene while maintaining gene expression under the control of endogenous regulatory elements.[6,49,50]. TAR cloning technology is selective and efficient and allows high-fidelity isolation of full-length genes with all of their regulatory elements (in these cases, the TAR-isolated human genes and their homologs were sequenced).[19,22,27,29,30,31] In perspective, TAR cloning engineering may provide a complete collection of full-length human genes (Figure 1) ready for CRISPR-mediated insertion or replacement with the following gene expression studies to elucidate the biological function of the diseaseassociated genes and to help with gene therapy for disease treatment

18 Molecular Therapy

Findings

Conclusions