RNA-Seq and WGS of CRISPRi strains based on Escherichia coli BW25993 during fosfomycin exposure.

The phosphonic antibiotic fosfomycin is a bacterial cell wall synthesis inhibitor, transporter loss or enzymatic inactivation confers resistance to fosfomycin. For the RNA-Seq, two Escherichia coli types were used: (1) CRISPRi library in YYdCas9: BW25993 CRISPRipgRNA intC::tetR-dcas9-aadA lacY::ypet-cat (Donati et al. 2021, https://doi.org/10.1016/j.cels.2020.10.011) and (2) YYdCas9: BW25993 intC::tetR-dcas9-aadA lacY::ypet-cat (BW25993 intC::tetR-dcas9-aadA lacY::ypet-cat araB::T7 RNAP-tetA DELTAaraB) Lawson et al., 2017. Bacteria were inoculated in LB medium and incubated at 220 rpm, 37C. Fosfomycin was added to a final concentration of 4x minimal inhibitory concentration (MIC) (304 ug/mL). Sequencing libraries were prepared with the Illumina Stranded Total RNA Prep kit, incorporating rRNA depletion with the Ribo-Zero Plus Microbiome Kit (Illumina). Libraries were then pooled and sequenced on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 (150 cycles). In order to find the genomic modification that caused fosfomycin resistance, whole genome sequencing (WGS) was performed on (1) an untreated control, (2) a sample treated for 9 hours with 4x MIC fosfomycin and (3) one sample treated for 24 hours. The full resistance was acquired after 24h. WGS libraries were prepared with the Illumina DNA Prep, (M) Tagmentation kit. Libraries were then pooled and sequenced on an Illumina iSeq 100 platform with sequencing run protocol 151,10,10,151. RNA extraction, RNA and DNA sequencing library preparation and NGS sequencing were performed at the Institute for Medical Microbiology and Hygiene (MGM) of the University of Tuebingen.

磷酰类抗生素磷霉素（fosfomycin）是一种细菌细胞壁合成抑制剂，细菌转运蛋白缺失或酶促失活可导致其对磷霉素产生耐药性。本研究的RNA测序（RNA-Seq）采用两种大肠杆菌（Escherichia coli）菌株：(1) 携带CRISPRi文库的YYdCas9菌株：BW25993 CRISPRipgRNA intC::tetR-dcas9-aadA lacY::ypet-cat（Donati等人，2021，https://doi.org/10.1016/j.cels.2020.10.011）；(2) YYdCas9菌株：BW25993 intC::tetR-dcas9-aadA lacY::ypet-cat（BW25993 intC::tetR-dcas9-aadA lacY::ypet-cat araB::T7 RNAP-tetA DELTAaraB）（Lawson等人，2017）。将细菌接种于LB培养基中，于37℃、220 rpm条件下振荡培养。随后向培养基中加入磷霉素，使其终浓度为4倍最低抑菌浓度（minimal inhibitory concentration, MIC）（304 μg/mL）。测序文库采用Illumina Stranded Total RNA Prep试剂盒构建，并搭配Ribo-Zero Plus Microbiome Kit（Illumina）进行核糖体RNA去除。将构建好的文库混合后，使用Illumina MiSeq平台及MiSeq Reagent Kit v3（150个循环）进行测序。为筛选导致磷霉素耐药的基因组变异，对三类样本进行全基因组测序（whole genome sequencing, WGS）：(1) 未处理的对照组样本；(2) 经4×MIC磷霉素处理9小时的样本；(3) 经4×MIC磷霉素处理24小时的样本。样本在处理24小时后获得完全耐药性。全基因组测序文库采用Illumina DNA Prep、(M) Tagmentation试剂盒构建。将文库混合后，使用Illumina iSeq 100平台，按照151,10,10,151的测序运行方案进行测序。RNA提取、RNA与DNA测序文库制备以及二代测序（next-generation sequencing, NGS）均在蒂宾根大学医学微生物学与卫生研究所（Institute for Medical Microbiology and Hygiene, MGM）完成。