Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
Mol Ther. 2021 Mar 3;29(3):1028-1046. doi: 10.1016/j.ymthe.2020.11.025. Epub 2020 Nov 26.
Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1,000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors, with each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions. Targeting efficiency was enhanced by target site transcription, while chromatin accessibility was associated with an increased likelihood of targeting. ChromHMM chromatin states characterizing transcription and enhancers in wild-type K562 cells were also associated with increased AAV-HR efficiency with and without target site transcription, respectively. Furthermore, the amenability of a site to targeting was influenced by the endogenous transcriptional level of intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.
重组腺相关病毒（rAAV）载体具有通过同源重组将转基因物靶向整合到特定基因组位点的独特能力，达到 0.1%-1%的频率。我们研究了影响大规模靶向效率的基因组参数。为此，我们在人类 HAP1 细胞系中生成了超过 1000 个工程化的、可诱导的、四环素诱导的靶位点，并使用携带独特条形码的 AAV-DJ 靶向载体文库感染了这个多克隆群体。每个靶位点的条形码整合的异质性提供了对该基因座靶向效率的评估。我们比较了相同染色体位置的靶位点转录和非靶位点转录的靶向效率。靶位点转录增强了靶向效率，而染色质可及性与靶向的可能性增加有关。在野生型 K562 细胞中，用于表征转录和增强子的 ChromHMM 染色质状态与有或无靶位点转录的 AAV-HR 效率增加也分别相关。此外，一个位点的靶向能力还受到相交基因的内源性转录水平的影响。这些结果定义了重要的参数，这些参数不仅可以帮助设计用于基因组编辑的最佳靶向载体，还可以为 AAV 介导的同源重组的机制提供新的见解。
