School of Physical Science and Technology, ShanghaiTech University, Shanghai, China.
Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Nat Chem Biol. 2024 Feb;20(2):180-189. doi: 10.1038/s41589-023-01420-4. Epub 2023 Sep 11.
CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.
CRISPR-Cas12f 核酸酶是目前最小的基因组编辑工具之一，由于其可以通过 cargo-size-limited adeno-associated virus 载体高效递送至细胞内，因此具有明显的优势。大多数已被鉴定的 Cas12f 核酸酶识别相似的富含 T 的原间隔序列邻近基序 (PAMs) 以实现 DNA 靶向，这大大限制了其靶向范围。在此，我们报道了一种新型的具有稀有 C 富含 PAM 特异性的微型 Clostridium novyi Cas12f1 核酸酶 (CnCas12f1，497 个氨基酸) 的低温电子显微镜结构和工程改造。结构特征揭示了详细的 PAM 识别、不对称同二聚体形成和单指导 RNA (sgRNA) 结合机制。sgRNA 工程改造将最初在细菌中无法进行基因组靶向的 CRISPR-CnCas12f1 转变为在人类细胞中有效的基因组编辑工具。我们的研究结果有助于进一步了解 CRISPR-Cas12f1 的工作机制，并扩展了微型 CRISPR 工具包。
