CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

中文翻译：

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CRISPR-Cas“非目标”位点抑制目标切割率


CRISPR-Cas 系统在真核和细菌系统的基因组编辑中已变得无处不在。 Cas9 与引导 RNA (gRNA) 形成复合物，并在 DNA 中搜索原型间隔子相邻基序 (PAM) 旁边的匹配序列（目标位点）。一旦找到，Cas9 就会切割 DNA。 Cas9 具有革命性意义，能够轻松改变 RNA 序列并靶向新位点。然而，虽然已经开发出预测 gRNA 特异性 Cas9 活性的算法，但仍缺乏对 gRNA 特异性活性的基本生物学理解。基因组中的 PAM 位点数量实际上是一个大量的抑制底物，与 Cas9/gRNA 复合物的靶位点竞争。我们证明，增加给定 gRNA 的非靶位点数量会以剂量依赖性方式降低靶向活性。此外，我们表明，使用对较小的 PAM 子集（或较小的竞争性底物池）具有更高的 PAM 特异性的 Cas9 突变体可以提高切割率，而增加 PAM 混杂性会降低切割率。通过增加 PAM 特异性来减少潜在搜索空间，为提高较慢的高保真 Cas9 变体的靶向活性提供了一条途径。通过工程改进 PAM 特异性来减少竞争性搜索空间，为工程化 Cas9 变体提供了一种替代策略，使其具有更高的特异性并保持了目标活性。