Fig. 2

In silico design of host 16S rRNA gene-specific gRNAs for Cas-16S-seq in rice. a The schematics of targeted DNA cleavage using Cas9 and gRNA. The 20 bp DNA target is paired with the gRNA guide sequence. A protospacer-adjacent motif (PAM, 5′-NGG-3′), which is indispensable for Cas9 binding, immediately follows the paired region. Red indicates the seed region that is less tolerance to mismatches for Cas9 binding. b The flowchart of the bioinformatics analysis procedure to evaluate gRNA specificities. The guide sequences of gRNA that target rice mitochondrial and chloroplast 16S rRNA gene (mt-gRNA and cp-gRNA) were extracted according to the requirements for Cas9/gRNA binding (Fig. 2a). Then, these guide sequences were aligned to prokaryotic 16S rRNA genes in the RDP database. Both NGG PAM and NAG PAM off-target sites in RDP-rRNA were considered here, although Cas9 weakly recognized NAG-PAM. The RDP-rRNA off-targets of each gRNA were identified using the criteria shown in the flowchart (see details in the “Methods” section). Finally, the total number of bacterial off-targets for each gRNA was used to rank the gRNA specificities (see Additional file 2: Table S1 and S2). c Number of mt-gRNAs and cp-gRNAs with different specificity ranks according to the number of RDP-rRNA off-targets. d Distribution of most specific mt-gRNA (< 10 off-targets) and cp-gRNA (< 1000 off-targets). The green boxes indicate hypervariable regions (V1–V9) of 16S rRNA gene. Gray boxes indicate the region of four 16S rRNA amplicons. The triangles indicate the gRNA position and are colored according to the number of RDP-rRNA off-targets. NA, not amplified