3. Results
3.1
Ahomozygous
strain of GOBPs deletion
A dual sgRNA-directed CRISPR-Cas9 system was employed to delete the
GOBPs genes. According to the genomic arrangement of GOBP1 and GOBP2
(Figure 1), the sgRNA1/2 and sgRNA3/4 were designed to target the
respective genes. The two specific sgRNAs and Cas9 protein were
co-injected into yellow peach moth early embryos, respectively. In
addition, to get both GOBP1 and GOBP2 (GOBP1/2) knockout homozygous
population, four specific sgRNAs (sgRNA1/2/3/4) with Cas9 protein were
using to co-inject. Among the injected eggs, 266 (96.72%), 180
(94.74%) and 199 (83.61%) neonates developed into adults
(G0) for GOBP1, GOBP2, and GOBP1/2, respectively (Table
1). Based on the PCR amplification results, the primer pair G1/2-F/R
revealed about 350 bp band (GOBP1) and about 600 bp band (GOBP2).
Sequencing of those
fragments
confirmed a deletion event of the genes that were created and inherited
in that single pair family. To get homozygous population, 10 homozygous
males and females (1:1) were put together to produce the next generation
(G1). Randomly extract DNA and sequenced using primer
pair G1/2-F/R from the G1, results showed all the samples have only one
expected band. To confirm those adults were homozygous, pair primers
T1-F/R and T2-F/R were used for testing. The results showed wild type
had one band, but not the same as knocked out GOBP1 and GOBP2 samples
(Figure 1). Sequencing the fragments of GOBP1 and GOBP2 confirmed each
gene had successfully knockout and the sequences consistent with our
expected (Figure 1). All homozygous adults keep generation and can be
used for further experiments.