RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Nat Biotechnol. 2022 Sep;40(9):1388-1393. doi: 10.1038/s41587-022-01255-9. Epub 2022 Apr 4.
Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein-protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.
尽管将基因组编辑工具进行拆分可以减小构建体的大小，但这需要特殊的工程技术将结合和催化结构域连接起来，因此，之前对于 Prime 编辑器 (PEs) 的递送和优化一直受到其较大的尺寸和复杂性的阻碍。我们在此报告了一种拆分的 PE（sPE），其中 Cas9 核酸酶（nCas9）与逆转录酶（RT）仍然未连接。sPE 在进行精确编辑方面的效率与亲本非拆分的 PE3 相似，并且没有增加插入缺失（indel）副产物。通过水动力注射将 sPE 递送到小鼠肝脏中以修饰β-连环蛋白，其驱动肿瘤形成的效率与 PE3 相似。使用两种腺相关病毒（AAV）载体递送可以纠正 I 型酪氨酸血症小鼠模型中的致病突变。同样，可以将 Prime 编辑引导 RNA（pegRNA）拆分为单个引导 RNA（sgRNA）和圆形 RNA RT 模板，以提高灵活性和稳定性。与之前的 sPE 相比，我们的 sPE 缺少内含子、蛋白-蛋白亲和模块和核酸酶敏感的 pegRNA 延伸，这增加了构建体的复杂性，并可能降低效率。我们的模块化系统将有助于 PE 的递送和优化。
