The log-phase cells of Escherichia coli MG1655 expressed I-C CRISPR-Cas and diverse SRU were harvested for RNA extraction. The total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, MA, USA) following the manufacturers protocol. Subsequently, purification was carried out using the phenol:chloroform method, followed by precipitation with an equal volume of ethanol. The purified RNA was stored at -80 C. For small RNA sequencing, 50 g RNA was subjected to treatment with T4 polynucleotide kinase (New England Biolabs, MA, USA) according to the manufacturers instructions at 37 C. The RNA was purified using the phenol-chloroform method and thenprecipitated with an equal volume of isopropanol and 0.1 volume of 3 M sodium acetate. The resulting RNA sample was re-dissolved in RNA-free water prior to sequencing. A small RNA library was constructed using the NEXTFLEX Small RNASeq Kit (Bioo Scientific, TX, USA), targeting RNA molecules ranging from 30 to 300 nt. The library was then subjected to Illumina HiSeq sequencing (paired-end, 150 bp reads). The obtained raw data reads underwent adapter removal and were mapped to the crlRNA sequence using previously reported Perl scripts.