Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC, USA.
Commun Biol. 2024 Jan 12;7(1):86. doi: 10.1038/s42003-024-05776-8.
As CRISPR effectors like Cas9 increasingly enter clinical trials for therapeutic gene editing, a future for personalized medicine will require efficient methods to protect individuals from the potential of off-target mutations that may also occur at specific sequences in their genomes that are similar to the therapeutic target. A Cas9 enzyme's ability to recognize their targets (and off-targets) are determined by the sequence of their RNA-cofactors (their guide RNAs or gRNAs). Here, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5' nucleotide extensions near its DNA-targeting segment-a modification that can increase gene editing specificity by orders of magnitude-to identify extended gRNAs (x-gRNAs) that effectively block any activity at those off-target sites while still maintaining strong activity at their intended targets. X-gRNAs that have been selected for specific target / off-target pairs can significantly out-perform other methods that reduce Cas9 off-target activity overall, like using Cas9 variants engineered for higher specificity in general, and we demonstrate their effectiveness in clinically-relevant gRNAs. Our streamlined approach to efficiently identify highly specific and active x-gRNAs provides a way to move beyond a one-size-fits-all model of high-fidelity CRISPR for safer and more effective personalized gene therapies.
随着 CRISPR 效应物（如 Cas9）越来越多地进入治疗性基因编辑的临床试验，未来的个性化医疗将需要有效的方法来保护个体免受潜在的脱靶突变的影响，这些突变也可能发生在与治疗靶点相似的基因组的特定序列中。Cas9 酶识别其靶标（和脱靶标）的能力由其 RNA 辅助因子（向导 RNA 或 gRNA）的序列决定。在这里，我们提出了一种方法，可以筛选数十万种带有短的、随机的 5'核苷酸延伸的 gRNA 变体，这些变体靠近其 DNA 靶向片段——这种修饰可以将基因编辑的特异性提高几个数量级——以识别扩展的 gRNA（x-gRNA），这些 gRNA 可以有效地阻止任何在这些脱靶位点的活性，同时仍然保持其预期靶点的强活性。针对特定靶标/脱靶对选择的 x-gRNA 可以显著优于其他降低 Cas9 脱靶活性的方法，例如使用通常针对更高特异性设计的 Cas9 变体，我们证明了它们在临床相关 gRNA 中的有效性。我们简化的方法可以有效地识别高度特异性和活性的 x-gRNA，为更安全、更有效的个性化基因治疗提供了一种超越一刀切的高保真 CRISPR 模式的方法。
