Koseki Sabrina, Hong Lauren, Yudistyra Vivian, Stan Teodora, Tysinger Emma, Silverstein Rachel, Kramme Christian, Amrani Nadia, Savic Natasha, Pacesa Martin, Rodriguez Tomás, Ponnapati Manvitha, Jacobson Joseph, Church George, Truant Ray, Jinek Martin, Kleinstiver Benjamin, Sontheimer Erik, Chatterjee Pranam
Duke University.
MIT Media Lab.
Res Sq. 2023 Mar 7:rs.3.rs-2625838. doi: 10.21203/rs.3.rs-2625838/v1.
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
CRISPR酶需要在引导RNA编程的靶位点侧翼有一个确定的原间隔相邻基序（PAM），这限制了它们在强大的基因组编辑应用中的序列可及性。在本研究中，我们将具有NRN>NYN PAM偏好的广泛靶向Cas9——SpRY的PAM相互作用结构域与具有同时广泛、高效和准确的NNG编辑能力的Cas9——Sc++的N端重组，以生成一种具有高度灵活PAM偏好的嵌合酶：SpRYc。我们证明SpRYc利用了这两种酶的特性来特异性编辑多种NNN PAM和疾病相关位点，用于潜在的治疗应用。总的来说，生成SpRYc的独特方法及其强大的灵活性，凸显了整合蛋白设计在Cas9工程中的力量，并推动了需要精确基因组定位的下游编辑应用。
