Programmed chromosome fission and fusion enable large-scale genome rearrangements and assembly

Up to this point, current genetic engineering tools have not been able to handle long stretches of DNA. Restriction enzymes, the standard tool for cutting DNA, can snip chunks of genetic material, and join the ends, to form small circular DNA segments that can be moved out of one cell and into another (if one tries this with stretches of linear DNA, survival is very short due to endonucleases that destroy linear DNA). Circular DNA can accommodate as much as several hundred thousand bases. However, synthetic biologists often want to move large segments of chromosomes containing multiple genes — which can be millions of bases in length (or more).

In addition, those cutting and pasting tools cannot be targeted precisely, which means that unwanted DNA can remain at the splicing sites — the equivalent of genetic scars. These errors can build up, as more changes are made. Another problem is that traditional editing tools cannot faithfully glue large segments together. Authors [see attached article & editorial] claim they have solved these problems. First, authors adapted CRISPR to excise precisely long stretches of DNA, without leaving scars. Then, authors altered another well-known tool — an enzyme called lambda red recombinase — so that it could glue the ends of the original chromosome (minus the removed portion) back together, as well as fuse the ends of the removed portion. Both circular strands of DNA are protected from endonucleases. This technique can create different circular chromosome pairs in other cells, and researchers can then swap chromosomes at will, eventually inserting (whatever chunk they choose) into the original genome.

Authors [see attached article & editorial] demonstrate the programmed fission [bacteria can duplicate its genetic material (DNA) and then divide into two, with each new organism receiving one copy of DNA) of the Escherichia coli genome into diverse pairs of synthetic chromosomes, plus the programmed fusion of synthetic chromosomes to generate genomes with user-defined inversions and translocations. Authors further combined genome fission, chromosome transplant, and chromosome fusion — in order to assemble genomic regions from different strains into a single genome. Thus, authors have programmed the scarless assembly of new genomes having nucleotide precision — a key step in the convergent synthesis of genomes from diverse progenitors. This work provides a set of precise, rapid, large-scale (megabase) genome-engineering operations for creating diverse synthetic genomes. These new tools will bolster industrial biotechnology by making it easier to vary levels of proteins that microbes make. 😊



· Science 30 Aug 2019; 365: 922-926 & editorial p. 849

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