目的·利用纳米孔Cas9靶向测序（nanopore Cas9-targeted sequencing，nCATS）技术实现金黄色葡萄球菌的快速检测，并同时进行葡萄球菌蛋白A（staphylococcal protein A，spa）分型和葡萄球菌盒式染色体mec（staphylococcal cassette chromosome mec，SCCmec）分型。方法·以spa基因和SCCmec基因元件作为靶向测序的2个目标区域（region of interest，ROI），设计4种CRISPR RNA（crRNA），使其构成的Cas9核糖核蛋白（ribonucleoprotein，RNP）能够靶向切割2个ROI两侧的序列；针对每种crRNA设计42 bp的模拟靶标DNA。通过Cas9 RNP切割效率测试，筛选合适的crRNA，优化切割体系，选择切割反应时间和Cas9 RNP合成温度。提取耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）菌株基因组DNA，末端去磷酸化，在切割后的ROI两侧末端加上dA尾，与测序接头连接后利用纳米孔机器进行测序。对测序结果数据的质量值进行分析，并将测得的核酸序列分别与mecA、spa和SCCmec数据库进行比对，根据比对结果判断样本中是否含有金黄色葡萄球菌，是否为MRSA，并同时获得其spa分型和SCCmec分型。结果·共设计出2组crRNA，经电泳图灰度分析，筛选出切割效率更高的一组用于后续实验。切割效率优化结果显示，Cas9 RNP与靶标DNA比例为1∶1，切割反应时间为15 min，Cas9 RNP合成温度为25 ℃时，Cas9 RNP切割效率可达87.41%。nCATS测序质量值集中在15（Q15）和20（Q20）之间，表明测序准确度约为99%。测序结果与mecA、spa和SCCmec数据库比对后得出菌株样本的spa分型为t2，并且是SCCmec Ⅱ型。相同菌株样本经PCR扩增后测序和多重PCR验证，结果均与nCATS结果一致。结论·利用nCATS技术能够实现金黄色葡萄球菌的快速检测，并能同时获得spa分型和SCCmec分型。

Objective ·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing (nCATS) technology, and simultaneously perform staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing. Methods ·The spa gene and SCCmec gene elements were selected as two regions of interest (ROIs) for targeted sequencing. Four types of CRISPR RNAs (crRNAs) were designed to form Cas9 ribonucleoproteins (RNPs) to cleave sequences flanking the two ROIs. For each crRNA, a 42 bp synthetic target DNA was designed. Appropriate crRNAs were screened, the cleavage system was optimized, and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing. Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus (MRSA) strain, and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed. Sequencing adapters were ligated, and sequencing was performed using a nanopore platform. The quality scores of the sequencing data were analyzed, and the obtained nucleic acid sequences were compared with those in the mecA, spa and SCCmec databases. Based on the comparison results, the presence of Staphylococcus aureus, MRSA or not, and spa and SCCmec types were determined. Results ·Two sets of crRNAs were designed. Based on grayscale analysis of electrophoresis results, the set with higher cleavage efficiency was selected for further experiments. Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA, a 15 min cleavage reaction time, and a Cas9 RNP synthesis temperature of 25 ℃ yielded a cleavage efficiency of 87.41%. nCATS sequencing quality scores ranged between 15 (Q15) and 20 (Q20), indicating an approximate sequencing accuracy of 99%. Sequence comparisons with the mecA, spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ. These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR. Conclusion ·The nCATS technique enables rapid detection of Staphylococcus aureus, while simultaneously providing spa and SCCmec typing information.