本期文章：《自然》：Online/在线发表

英国MRC分子生物学实验室Jason W. Chin研究组实现大肠杆菌基因组片段的连续合成和Mb级人类DNA组装。2023年6月28日，《自然》杂志在线发表了这项成果。

研究人员开发了细菌人工染色体（BAC）逐步插入合成法（BASIS），这是一种在大肠杆菌外显子中进行百万级DNA组装的方法。研究人员用BASIS组装了1.1Mb的人类DNA，其中含有大量的外显子、内含子、重复序列、G-四聚体和长短穿插核元件（LINE和SINE）。BASIS为构建不同生物体的合成基因组提供了一个强大的平台。研究人员还开发了连续基因组合成（CGS），这是一种用合成DNA连续替换大肠杆菌基因组的100 kb片段的方法；CGS最大限度地减少了合成DNA和基因组之间的交叉，从而使每个100 kb替换的输出无需测序即可为下一个100 kb替换提供输入。

使用CGS，研究人员在10天内从5个外显子中合成了大肠杆菌基因组的0.5Mb部分，其全部合成的一个关键中间环节。通过平行化CGS并将其与快速寡核苷酸合成和外显子组组装结合起来，再加上从带有不同合成基因组部分的菌株中汇编单一基因组的快速方法，研究人员预计将有可能在不到2个月的时间内从功能设计中合成整个大肠杆菌基因组。

据了解，全基因组合成为理解和扩展生物体的功能提供了一个强大的方法。为了快速、可扩展和并行地构建大基因组，人们需要（1）从较短的前体组装兆字节DNA的方法和（2）用合成DNA快速和可扩展地替换生物体基因组DNA的策略。

附：英文原文

Title: Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly

Author: Zrcher, Jrme F., Kleefeldt, Askar A., Funke, Louise F. H., Birnbaum, Jakob, Fredens, Julius, Grazioli, Simona, Liu, Kim C., Spinck, Martin, Petris, Gianluca, Murat, Pierre, Rehm, Fabian B. H., Sale, Julian E., Chin, Jason W.

Issue&Volume: 2023-06-28

Abstract: Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1,2,3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)—a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)—a method for continuously replacing sequential 100kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100kb replacement provides, without sequencing, the input for the next 100kb replacement. Using CGS, we synthesized a 0.5Mb section of the E. coli genome—a key intermediate in its total synthesis1—from five episomes in 10days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2months.

DOI: 10.1038/s41586-023-06268-1