CRISPR RNA preparation

We designed the CRISPR RNA (crRNA) using in-silico analysis and the basic local alignment search tool (BLAST) to specifically detect the lipL32 gene sequence adjacent to the TTTN protospacer adjacent motif (PAM) site. The lipL32 crRNA sequence was 5′-UAAUUUCUACUAAGUGUAGAUUUCUGAGCGAGGACACAAUC-3’, consisting of a scaffold 5′-UAAUUUCUACUAAGUGUAGAU-3’ and a guide RNA 5′-UUCUGAGCGAGGACACAAUC-3’, both of which were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, UK). For the preparation of crRNA, synthetic oligonucleotides were ordered as ultramer DNA (Macrogen, South Korea) with an appended T7 promoter sequence. Oligonucleotides for crRNA (1 μM) were annealed to a short T7 primer (final concentration of 10 μM each) and incubated with T7 polymerase at 37°C for 2 h. The crRNA was then purified using a Monarch RNA Cleanup Kit (New England Biolabs, UK). The concentration of purified crRNA product was measured using a Qubit miRNA assay kit and Qubit 4 Fluorometer (Thermo Scientific, USA) and stored at -80°C until further use.