Targeted mitosequencing of genomic DNA using CRISPR Cas9: ‘targeted mitosequencing’
Design of Cas9 guide RNAs: The CRISPR Cas9 enzyme cleaves at specific sites of your choice with the aid of the so-called guide RNA (gRNA) sequences. The latter were designed based on an alignment of several salmonid species and rockfish species that were of interest and sequences compatible to all the species were found using the ‘Find CRISPR Site’ tool within Geneious Prime 2021.2.2 (Biomatters Ltd, www.geneious.com). This tool searches for sequences of 20 nucleotide followed by a protospacer adjacent motif (PAM) on the 3’ end of three nucleotides with the motif NGG. Five candidate gRNAs (Table1, S1) were selected to ensure there was going to be enough coverage in the event of one or two failing due to mismatches as well as to ensure even coverage (López-Girona et al., 2020). These cutting sites are conserved regions of the mitogenome in fishes.: one (alias 41) on the 12SrRNA gene (12S), two (alias 28 and 17) about 2 kbp further on the 16SrRNA (16S) and two more are overlapping and cleave on the tRNA-Gly (G) that is located between the COIII and ND3 coding genes (alias 20 and 24) about 7 kbp away. The cutting sites on the tRNA-Gly were chosen to increase the coverage at approximately halfway on the mitogenome from the 16S or 12S cuts to ensure the (cytochrome b) cytb and (control region) D-loop areas were covered evenly in which seemed a promising site although there was no guarantee of its match with most of the fish due to lack of representation of this gene in GenBank. The complete mitogenome of 2941 specimens was downloaded and the specificity of the gRNAs was tested in silico against the consensus of the alignment of 176 full Chondrichthyes mitogenomes (with 0,0, 0, 5, 5 mismatches for gRNA alias 41, 28, 17, 20 and 24, respectively), 13 Cyclostomata (2, 2, 0, 5, 5), 7 Sarcopterygii (0, 0, 0, 3, 3) and 2693 Teleostei (0, 0, 0, 2,2). These five gRNA were also tested against the whole genome of Sebastes schlegelii(GenBank Acc ASM1467356) to assess whether the guides could target nuclear regions using a Bowtie2 alignment within Geneious, with low sensitivity/fast parameters and no contigs were found The gRNA sequences were synthesized with a crRNA tail that will bind to the transactivating RNA (tracRNA) to form the duplex that the Cas9 uses to find the cutting sites (IDT, Skokie, IL, USA).
Table 1. Details of the guide RNA (gRNA) sequences used to direct the CRISPR-Cas9 scission. The gRNA positions (5’-3’) are based on the mitogenome sequence of Sebastes aleutianus (GenBank Acc. NC039779).