We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7–10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR–Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.

我们之前开发了REXER（Replicon EXcision Enhanced Recombination）；这种方法可以在一个步骤中用合成 DNA替换大于 100 kb 的大肠杆菌基因组，并允许通过核苷酸分辨率快速识别无活力或其他有问题的序列。REXER 的迭代重复（GENESIS，GENomE Stepwise Interchange Synthesis）可以用合成 DNA 逐步替换较长的连续基因组 DNA 部分，甚至替换整个大肠杆菌带有合成 DNA 的基因组。在这里，我们详细介绍了 REXER 和 GENESIS 的协议。标准的 REXER 协议通常需要 7-10 天才能完成。我们的描述包括 (i) 合成 DNA 设计，(ii) 合成 DNA 构建体的组装，(iii) 利用 CRISPR-Cas9 与 lambda-red 重组和正/负选择相结合，以实现基因组 DNA 的高保真替换DNA（或合成 DNA 的插入），(iv) 评估整合和替换的成功和 (v) 用核苷酸分辨率鉴定非耐受合成 DNA 序列。该协议提供了一套精确的基因组工程方法来创建定制的合成大肠杆菌基因组。