Megabase-scale human genome rearrangement with programmable bridge recombinases

Bridge recombinases are naturally occurring RNA-guided DNA recombinases that we previously demonstrated can programmably insert, excise, and invert DNA in vitro and in bacteria. Here, we report the discovery and engineering of the bridge recombinase ortholog ISCro4 for universal rearrangements of the human genome. We define strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the ISCro4 bridge RNA and deep mutational scanning of its recombinase, we achieve up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrate intra-chromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Finally, we provide proof-of-concept for excision of disease-relevant gene regulatory regions or expanded repeats.

桥接重组酶（Bridge recombinases）是天然存在的RNA引导型DNA重组酶（RNA-guided DNA recombinases），此前我们已证实其可在体外（in vitro）及细菌体内对DNA实现可编程的插入、切除与倒位。本研究报道了可用于人类基因组全域重排的桥接重组酶同源物（ortholog）ISCro4的发现与工程化改造。我们明确了桥接系统的最优应用策略，并借助机制层面的认知提升其靶向特异性。通过对ISCro4的桥接RNA进行理性工程化改造，以及对其重组酶开展深度突变扫描（deep mutational scanning），我们实现了最高达20%的人类基因组插入效率，以及最高82%的全基因组特异性（genome-wide specificity）。我们进一步证实了染色体内倒位与切除操作，可介导长达0.93兆碱基的DNA片段的转移。最后，我们针对疾病相关基因调控区域或扩增重复序列的切除提供了概念验证。