The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

广泛使用的化脓性链球菌Cas9 (SpCas9) 核酸酶的 DNA 靶向特异性源自蛋白质-DNA 与原型间隔子相邻基序 (PAM) 序列的接触，以及其引导 RNA 和靶 DNA 之间的碱基配对相互作用。之前的报告已经证实，SpCas9 的 PAM 特异性可以通过定向进化或其他蛋白质工程策略的正选择程序来改变。在这里，我们利用体内定向进化系统，该系统结合了同时的正向和负向选择来进化SpCas9变体，相对于野生型，其对NAG PAM的活性相当或提高，并且对NGG PAM（特别是YGG PAM）的活性降低。我们还表明，可用进化中间体的 PAM 偏好有效地决定了类似的反选择 PAM 是否会引发不同的选择严格性，并证明通过补偿性锌指与 SpCas9 的融合，可以在酵母选择系统中特异性地增加负选择。