Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1M1, Canada.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Cell Rep. 2019 Nov 12;29(7):1739-1746.e5. doi: 10.1016/j.celrep.2019.10.017.
CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.
CRISPR-Cas9 系统为基因组编辑提供了强大的工具。然而，要优化这项技术的应用，就需要控制 Cas9 的活性，以便精确调节编辑的时间、组织特异性和准确性。抗 CRISPR 蛋白是一种天然存在的 CRISPR-Cas 系统抑制剂，体积小，非常适合这一目的。已经有许多抗 CRISPR 蛋白被证明可以强烈抑制 CRISPR-Cas9 系统的亚群，但它们的最大抑制活性通常仅限于特定的 Cas9 同源物。由于 Cas9 同源物在重要特性上存在差异，不同的 Cas9 可能是特定基因组编辑应用的最佳选择。为了促进多种 Cas9 同源物的实际应用，我们在这里鉴定了一种叫做 AcrIIA5 的抗 CRISPR 蛋白，它可以强烈抑制九种不同的 II-A 型和 II-C 型 Cas9 同源物，包括目前用于基因组编辑的 Cas9 同源物。我们表明，AcrIIA5 的活性导致单引导 RNA(sgRNA)的部分体内切割，这表明其机制涉及 RNA 相互作用。
