REXER & GENESIS
In order to expand the capacity to handle DNA to 100-kb scale in a single step, we invented the Replicon Excision Enhanced Recombination (REXER) method and the Genome Stepwise Interchange Synthesis (GENESIS) strategy1. In a single integrated step, REXER cuts open the old genomic sequence using CRISPR/Cas9 activity, rejoins the double strand breaks (DSB) with 50-bp homology arms that leads to the replacement of the wildtype fragment, and selects clones with correctly integrated synthetic DNA using combinations of positive (+1, +2) and negative (-1, -2) selection markers (Fig. 1a).
As a result, REXER enables highly efficient and accurate one-step in vivo genome integration of large synthetic DNA fragment (Fig. 1b, c)1. The efficiency of REXER is independent of the DNA size to be integrated (Fig. 1b)1. Thus, the size capacity of each REXER step is only restricted by the ability to deliver synthetic DNA into E. coli cells (currently at ~100 kb for electroporation). Iterations of REXER through the GENESIS strategy allow for stepwise replacement of even larger fragment of the genome, and ultimately the de novo synthesis of the entire E. coli genome (Fig. 1d). Recently, I have lead the collaborated effort in streamlining the REXER and GENESIS protocol2 from assembly of 10-kb synthetic DNA to 100-kb fragments in yeast, integration of 100-kb by REXER, step-wise iteration by GENESIS, and identification of design flaws through NGS and sequence analysis. The streamlined protocol of REXER takes minimally seven days for one iteration2, and approximately two weeks if trouble-shooting of design flaws is required2.
Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514–518 (2019).