Enhancing the Ministring DNA (msDNA) Purification Using PI-Sce1 Homing Endonuclease/CRISPR-Cas3 Recombinant System

Abstract

In the generation of msDNA the recombinant E. coli cells are transformed by a msDNA generating precursor plasmid, whereupon expression of the Tel protelomerase enzyme, acting on the pal target sequence present in the precursor plasmid, generated linear covalently closed (LCC) msDNA. However, the in vivo recombinant platform to produce msDNA results in a mixture of plasmids including unprocessed precursor plasmid, unwanted LCC bacterial backbone, and their topological isoforms, which interferes with the purification of the target species. For larger scale synthesis, the plasmid extract needs to be pretreated with commercially available restriction enzymes before being purified through chromatographic columns. Meanwhile, at the laboratory scale, msDNA is purified from agarose gels based on their size. These purification processes are time-consuming and inefficient and therefore, there is a need to optimize the process. To address this issue, we developed two in vivo recombinant systems for digesting the unwanted prokaryotic backbone and unprocessed precursor plasmid. These systems are the PI-SceI homing endonuclease enzyme system and the clustered regularly interspaced short palindromic repeats-Cas3 (CRISPR-Cas3) system. Homing endonucleases are highly specific DNA cleaving enzymes. The homing endonuclease PI-SceI, encoding gene vma from Saccharomyces cerevisiae was successfully integrated into the tel integrated bacterial chromosome via site-specific recombination using conditional replication and integration (CRIM) plasmid. The double integrants, both vma and tel integrated recombinant bacteria, were transformed with msDNA synthesizing precursor plasmids and induced the msDNA synthesis and vma gene overexpression. Even though the double integrants were able to overexpress the homing endonuclease enzyme and digest the precursor plasmid, they were not able to synthesize msDNA. Therefore, the Tel protelomerase enzyme was expressed episomally inside the vma integrated recombinant bacteria. This vma gene is under the control of an inducible PBAD promoter. In the presence of L-arabinose in the media, the Tel protelomerase enzyme was episomally expressed and synthesized msDNA by acting on the precursor plasmid. Subsequently, the overexpressed PI-SceI homing endonuclease enzyme digested the undesired byproducts of msDNA synthesis as expected. Introducing homing endonuclease enzyme recognition sequences into the Tel protelomerase enzyme-expressing plasmid will further improve the purification process. The other recombinant system that was developed is the utilization of the CRISPR-Cas3 system which is naturally present in W3110 E. coli K-12 bacteria. A pre-crRNA targeting the origin of replication (ori) of the msDNA synthesizing precursor plasmid was successfully designed and cloned into the low copy number plasmid. The pre-crRNA expressing gene cassette was placed under the control of the PBAD promoter. Upon overexpression, crRNA was synthesized inside the W3110 E. coli K-12 bacteria. The crRNA bound to the expressed CRISPR-Cas3 protein cascade of the bacteria, guided the effector complex to the target sequence and successfully digested the targeted precursor plasmid. Even though the W3110 tel+ recombinant bacteria synthesized msDNA in a pre-crRNA expressing background, an efficient degradation of the unwanted by-products of msDNA synthesis was not observed. This could be due to the disruption of the CRISPR locus of W3110 tel+ recombinant bacteria. Episomal expression of the CRISPR-Cas genes inside W3110 tel+ recombinant bacteria will enhance the digestion of the non-msDNA species.