Boyle Evan A, Becker Winston R, Bai Hua B, Chen Janice S, Doudna Jennifer A, Greenleaf William J
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Program in Biophysics, Stanford University, Stanford, CA 94305, USA.
Sci Adv. 2021 Feb 19;7(8). doi: 10.1126/sciadv.abe5496. Print 2021 Feb.
The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to "proofreading" capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.
RNA引导的核酸酶Cas9开启了强大的方法，可通过靶向DNA切割来扰动基因组，以及通过靶向DNA结合来扰动调控组，但有限的生化数据阻碍了对不同引导序列间靶标结合和切割的序列扰动进行定量建模的努力。我们提出了基于测序的可扩展平台，用于高通量过滤结合和切割，然后对90个装载有不同引导RNA的Cas9核糖核蛋白（RNP）的35,047个靶向和脱靶DNA序列进行了62,444次定量结合和切割测定。我们观察到，不同RNP的结合和切割效率以及特异性差异很大；经典研究的引导序列通常具有非典型的高特异性；靶标周围的序列背景调节Cas9的结合速率；并且Cas9 RNP可能将靶标隔离在无生产性的状态，这有助于“校对”能力。最后，我们将研究结果提炼成一个可解释的生物物理模型，该模型可预测不同靶标序列扰动下结合和切割的变化。
