During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology—termed a multitron—for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.

近年来，在 CRISPR 向导 RNA 上支架上的文库规模基因组操作的使用具有变革性。然而，这些现有方法通常是跨基因组的多重检测。不幸的是，构建具有多个不相邻精确突变的细胞仍然是一个费力的编辑循环，隔离编辑后的细胞并再次编辑。使用细菌逆转录素可以克服这一限制。Retron 是由逆转录酶和非编码 RNA 组成的遗传系统，其中包含多拷贝单链 DNA，该 DNA 经过逆转录可产生单链 DNA 的多个拷贝。在这里，我们描述了一种称为 multitron 的技术，该技术使用 retron 阵列同时精确修饰单个基因组上的多个位点，其中从单个转录本产生多个供体编码 DNA。多子结构与原核细胞中的重组和真核细胞中的 CRISPR 编辑兼容。我们展示了这种方法在分子记录、遗传元件最小化和代谢工程中的应用。