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).