Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

Abstract

Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology.

Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning‐free workflows

“…Thus, ZEP genes may have been expressed in the transformed cells causing toxicity. Another plausible cause is related to the toxicity of the DNA itself (Kouprina & Larionov, 2016).…”

“…Using this approach, an assembly of up to nine fragments in a 21-kb vector was carried out using 60-bp overlap regions (Kuijpers et al, 2013). Furthermore, so far this method has been shown to be much more effective for dealing with large DNA fragments than traditional cloning in Escherichia coli (Kouprina & Larionov, 2016). For instance, due to instability issues of large DNA constructs in E. coli, in vivo DNA assembly in yeast was critical for assembling the first synthetic bacterial genomes (Gibson, Benders, Andrews-Pfannkoch, et al, 2008;Gibson, Benders, Axelrod, et al, 2008;Gibson et al, 2010).…”

Synthetic Genomics: From DNA Synthesis to Genome Design

“…Gateway, InFusion TM , uracil‐specific excision reagent (USER) cloning, sequence and ligation independent cloning (SLIC), circular polymerase extension cloning (CPEC), and Gibson assembly are sequence‐independent overlap methods that are better options for larger polynucleotide assemblies. In contrast to the in vitro assembly methods described above, transformation‐associated recombination (TAR) in S. cerevisiae , the “domino method” in Bacillus subtilis, and Cas9‐facilitated homologous recombination assembly (CasHRA) are in vivo cloning methods that take advantage of powerful DNA recombination systems and enable the assembly of megabase‐sized genomes. The choice of DNA assembly method is largely a matter of preference, and multiple approaches are often applied sequentially to achieve the final goal.…”

Synthetische Genomik: von der DNA‐Synthese zu Designer‐Genomen

“…BglBricks, [6] BioBricks, [7] Golden Gate [8] und MASTER-Ligation (methylation-assisted tailorable ends rationale) [9] sind Assemblierungstechniken auf der Basis von Restriktionsenzymen und weit verbreitet fürd ie standardisierte Assemblierung von biologischen Komponenten. Im Unterschied zu den oben beschriebenen In-vitro-Assemblierungsmethoden handelt es sich bei der transformationsassoziierten Rekombination (TAR) [51] in S. cerevisiae,d er "Dominomethode" in Bacillus subtilis [16] und CasHRA (Cas9facilitated homologous recombination assembly) [17] um Invivo-Klonierungstechniken, die sich effiziente DNA-Rekombinationssysteme zunutze machen und die Konstruktion von Genomen im Megabasenbereich ermçglichen. Im Unterschied zu den oben beschriebenen In-vitro-Assemblierungsmethoden handelt es sich bei der transformationsassoziierten Rekombination (TAR) [51] in S. cerevisiae,d er "Dominomethode" in Bacillus subtilis [16] und CasHRA (Cas9facilitated homologous recombination assembly) [17] um Invivo-Klonierungstechniken, die sich effiziente DNA-Rekombinationssysteme zunutze machen und die Konstruktion von Genomen im Megabasenbereich ermçglichen.…”

“…Gateway, [10] InFusion, [11] USER-Klonierung (uracil-specific excision reagent), [12] SLIC (sequence and ligation independent cloning), [13] CPEC (circular polymerase extension cloning (CPEC) [14] und Gibson-Assemblierung [15] sind sequenzunabhängige Überlappmethoden, die bessere Optionen fürd ie Assemblierung grçßerer Polynukleotide darstellen. Im Unterschied zu den oben beschriebenen In-vitro-Assemblierungsmethoden handelt es sich bei der transformationsassoziierten Rekombination (TAR) [51] in S. cerevisiae,d er "Dominomethode" in Bacillus subtilis [16] und CasHRA (Cas9facilitated homologous recombination assembly) [17] Genoms von M. genitalium JCVI-1.0 In-vitro-Gibson-Assemblierung,S tandardklonierung in E. coli und TAR-Assemblierung in Hefe genutzt. [18]…”