CRISPR/Cas9 Mediated Genome Editing In Lamprey And Zebrafish

Recently, biotechnology like ZFN(Zinc-finger nucleases), TALEN(Transcription activator-like effector nucleases) and CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) has been used in editing genome on cells and organisms. Among these biotechnologies, CRISPR/Cas9 system became popular for its simplicity and efficiency.Lamprey is a kind of agnathans, or jawless vertebrate, and it has been taken as a living fossil of biological evolution. Studies on lamprey contribute to uncover the mechanism of vertebrate development and original of specific feature existed in vertebrate, such as neural crest, hindbrain, pharyngeal, jaw, spinal cord and appendage. The traditional way of studying function genes is to acquire targeted homozygous mutant by inherited from a chimera, then further study the gene function using the mutant. In this research, our research material was the Northeast Chinese lamprey(Lethenteron morii), which was a kind of aquatic organism lived in northeast area of China under low temperature. The northeast lamprey had to go through several metamorphosis stages before sex mature, which had a high requirement for water quality. As the complicated biological habits impeded artificial breeding in the laboratory, functional genes study on lamprey was greatly restricted.In this study, we constructed two lamprey codon optimized cas9 plasmids to knock out golden(gol,slc24a5), kctd10, wee1, soxe2 and wnt7 b five genes. The results showed that all these five genes had been successfully knock out by T7E1 enzyme digestion test and PCR products sequencing. To further verify the high efficiency of mutant, we sequenced the injected embryos’ target genome, and the sequence results showed the percentage of mutation were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively.We found the embryos injected with gol Cas9-gRNA or kctd10 Cas9-gRNA caused regularly mutator phenotype by morphologic observation. The gol encoded the sodium/calcium exchanger Slc24a5 that affects pigmentation in zebrafish. As the gol was mutant in zebrafish, the amount of melanophore on the skin and retina would reduce. Therefore, it was a convenient way to estimate the mutation of biallelic alleles. For the other gene, kctd10, which is a member of the potassium channel tetramerization domain(KTTD)-containing family. Mutation of kctd10 in zebrafish resulted malformation of the atrioventricular canal, causing a stretched and string-like heart, which could be easily read out after 36 hours post fertilization( hpf).We counted the amount of injected lamprey of gol and kctd10, and the percentage mutation rate were 38.6% and 85.6%, respectively. While the results gotten from T7E1 enzyme digestive and Monoclonal sequencing both revealed that the gol had a high target gene mutation. We found that the target site of gol was located in a less conserved region of genome when we blast the homologous gene in human species, mouse and zebrafish. On the contrast, the kctd10 was located in a conserved region of genome. So we speculate that choosing a target in a functional conservative region would increase the phenotypic mutation. In all, we constructed two lamprey codon optimized Cas9 plasmid that have high efficiencies in knocking out target genes and make recessive null-like phenotypes appeared in F0. We believed that the CRISPR/Cas9-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches.Hox gene is a group of transcript factor family, which control the body construction in embryonic development. The mutations of Hox gene often lead to the malformation of early embryos. The genome of invertebrate often has only one Hox gene cluster, while the vertebrate has at least 4 Hox clusters in the genome as the double duplication of the whole genome during the biological evolution. The zebrafish, one kind of teleosts, which has go through another chromesome duplication, finally shaped 7 Hox clusters as for the deletion or mutation of genome during the evolutionI successfully edited the zebrafish Hox gene(cluster) with CRISPR/Cas9 system. On one hand, I knock out the target site of HoxA2 b,HoxB2a,HoxB7 a,HoxB10a up,HoxB10 a down,HoxB13 a,HoxC1a,HoxC3 a,HoxC12b and HoxC13 b. The efficiency of mutant is range from 20% to 90%. On the other hand, I deleted the fragment of HoxB10 a, HoxBa cluster, HoxBb cluster and HoxCb cluster and the ratio of the heritable were 2/40, 1/30, 3/40 and 0/90, respectively. We found the efficiency of single site mutant was high than that of the deletion of segment, whereas we did not find the correspondence between the deletion length of genome part and the efficiency of deletion for the lack of ample samples. I think we could have a better understanding of the functional differentiation of Hox gene clusters duplication and the specific Hox genes function in teleosts by further study for these Hox gene(cluster) homozygous mutation.