Bioproduction Research Institute, The National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Inplanta Innovations Inc., Yokohama, Japan.
CRISPR J. 2024 Aug;7(4):197-209. doi: 10.1089/crispr.2024.0013. Epub 2024 Aug 7.
The genome-editing efficiency of the CRISPR-Cas9 system hinges on the recognition of the protospacer adjacent motif (PAM) sequence, which is essential for Cas9 binding to DNA. The commonly used (SpyCas9) targets the 5'-NGG-3' PAM sequence, which does not cover all the potential genomic-editing sites. To expand the toolbox for genome editing, SpyCas9 has been engineered to recognize flexible PAM sequences and Cas9 orthologs have been used to recognize novel PAM sequences. In this study, Cas9 (AalCas9, 1059 aa), which is smaller than SpyCas9, was found to recognize a unique 5'-NNACR-3' PAM sequence. Modification of the guide RNA sequence improved the efficiency of AalCas9-mediated genome editing in both plant and human cells. Predicted structure-assisted introduction of a point mutation in the putative PAM recognition site shifted the sequence preference of AalCas9. These results provide insights into Cas9 diversity and novel tools for genome editing.
CRISPR-Cas9 系统的基因组编辑效率取决于对原间隔基序 (PAM) 序列的识别，这对于 Cas9 与 DNA 的结合至关重要。常用的 (SpyCas9) 靶向 5'-NGG-3' PAM 序列，但它不能涵盖所有潜在的基因组编辑位点。为了扩展基因组编辑工具包，SpyCas9 已被设计为识别灵活的 PAM 序列，并且已经使用 Cas9 同源物来识别新的 PAM 序列。在这项研究中，发现比 SpyCas9 更小的 AalCas9（1059 个氨基酸）识别独特的 5'-NNACR-3' PAM 序列。对向导 RNA 序列的修饰提高了 AalCas9 在植物和人类细胞中介导的基因组编辑效率。在假定的 PAM 识别位点的预测结构辅助引入点突变改变了 AalCas9 的序列偏好。这些结果为 Cas9 多样性提供了深入了解，并为基因组编辑提供了新工具。
