Schematic diagram of Cas-16S-seq. Cas-16S-seq is based on the regular 16S-seq procedure using two-step PCR to amplify and index 16S rRNA genes. In the first PCR, universal primers with adaptors (Rd-universal-F/R) are used to amplify the variable regions of 16S rRNA genes. Then, Cas9 and specific gRNA is used to cleave the host 16S rRNA genes in the PCR product. In the second index PCR, primers containing index and Illumina sequencing adaptors (P5-index-Rd-F and P7-index-Rd-R) are used to amplify the digested products and obtain the final amplicon library. The host 16S rRNA gene fragments are not amplified in the second PCR and are therefore removed in the final 16S-seq data.