通过双gRNA进行可预测的高效基因编辑

CRISPR基因编辑是通过使用内源DNA修复机制修复Cas9引入的双链断裂（DSB）来完成的。尽管对于给定的CRISPR指导RNA（gRNA），DSB修复的结果是可预测的，但在不同的靶位点之间它是不同的。在这里，我们报道了由Cas9和一对gRNA引入的缺失连接是可预测的发现。我们研究了在三个人类细胞系中靶向四基因（TTR，CREB1，STAT2和IRF9）的8对gRNA的修复连接。使用Sanger和二代测序和ICE分析，我们发现缺失连接主要由非同源平末端连接（NHBEJ）修复。我们进一步评估了24个CRISPR缺失接头的插入缺失谱，以证实这一观察结果，并进一步证明了CRISPR对之间的PAM方向和距离对NHBEJ结果有轻微影响。基于这些观察结果，我们应用NHBEJ策略引入了高度精确且可预测的基因敲除和DMD外显子51的框内缺失，为杜兴氏肌营养不良症和许多其他人类疾病的CRISPR基因治疗提供了一种新颖的替代治疗策略。

CRISPR gene editing is accomplished by utilizing endogenous DNA repair mechanisms to repair double-strand breaks (DSBs) introduced by Cas9. Although the outcome of DSB repair is predictable for a given CRISPR guide RNA (gRNA), it differs across distinct target sites. Here, we report the finding that deletion junctions induced by Cas9 and a pair of gRNAs are predictable. We examined repair junctions of 8 gRNA pairs targeting four genes (TTR, CREB1, STAT2 and IRF9) in three human cell lines. Using Sanger sequencing, next-generation sequencing and ICE analysis, we discovered that deletion junctions are primarily repaired via non-homologous blunt-end joining. We further evaluated the indel profiles of 24 CRISPR deletion junctions to validate this observation, and additionally demonstrated that the PAM orientation and distance between the two gRNAs exert a subtle impact on the outcomes of non-homologous blunt-end joining. Based on these findings, we applied the non-homologous blunt-end joining strategy to generate highly precise and predictable gene knockouts and in-frame deletions of DMD exon 51, offering a novel alternative therapeutic approach for CRISPR-based gene therapy of Duchenne muscular dystrophy and many other human genetic diseases.