Structural basis of Faecalibaculum rodentium Cas9 with high genome editing efficiency and fidelity provide insights into CRISPR-Cas protein engineering

Bacteria Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits efficient cleavage activity and high genome editing fidelity by recognizing the 5'-NNTA-3' protospacer adjacent motif (PAM) sequence. This system may have an advantage in targeting the TATA box within eukaryotic promoters. However, the structural information of FrCas9 is largely unknown. Herein, we report two cryo-electron microscopy structures of FrCas9:sgRNA:DNA complex capturing the R-loop expansion and pre-catalytic status, at 2.89 angstrom and 3.6 angstrom resolution respectively. These complex structures elucidate the unique recognition patterns of the 5'-NRTA-3' PAM and sgRNA:DNA heteroduplex, highlighting specific key residues in FrCas9 nuclease activity and fidelity. We confirmed that the phosphate lock loop region of FrCas9 protein potentially enhances the tugging effects on the target-strand phosphate between the PAM and PAM-proximal +1 base pair, facilitating R-loop nucleation through the introduction of linear positively charged side chain residue substitutions. The genome-wide unbiased identification of DNA double-stranded breaks enabled by sequencing (GUIDE-seq) assays results show that FrCas9 phosphate lock loop variant V1103K improved the overall fidelity on 9 gene targets by up to 32.25%. Combined with alternations of heteroduplex interacting residues, a series of structural-based designed FrCas9 variants display synergistic effects on editing efficiency and fidelity in human cells. Together, our experiments provide the most detailed structural information for FrCas9 to date, and present the mechanism framework of a high fidelity Cas9 member with potential broadly applications.

啮齿类粪便杆菌（Faecalibaculum rodentium，简称Fr）的CRISPR-Cas9系统可通过识别5'-NNTA-3'原型间隔序列毗邻基序（protospacer adjacent motif，PAM），展现出高效的切割活性与极高的基因组编辑保真度。该系统在靶向真核启动子内的TATA盒时具备独特优势。然而，目前学界对FrCas9的结构信息尚知之甚少。本研究报道了两种分别捕获R环扩张状态与预催化状态的FrCas9:单向导RNA（single guide RNA，sgRNA）:DNA复合物的冷冻电镜结构，分辨率分别为2.89埃与3.6埃。上述复合物结构阐明了FrCas9对5'-NRTA-3' PAM以及sgRNA:DNA异源双链的独特识别模式，揭示了与FrCas9核酸酶活性和保真度相关的关键特异性残基。本研究证实，FrCas9蛋白的磷酸锁环（phosphate lock loop）区域可通过引入带正电的线性侧链残基替换，增强其对PAM与PAM近端+1碱基对之间靶链磷酸基团的牵拉效应，进而促进R环成核。基于测序的全基因组无偏DNA双链断裂识别技术（GUIDE-seq）实验结果显示，FrCas9的磷酸锁环突变体V1103K可使9个基因靶点的整体编辑保真度提升最高达32.25%。结合异源双链相互作用残基的改造，一系列基于结构设计的FrCas9突变体在人类细胞中展现出编辑效率与保真度的协同提升效应。综上，本研究为FrCas9提供了迄今为止最为详尽的结构信息，并阐明了一类具备广阔应用潜力的高保真Cas9家族成员的作用机制框架。