Progress 08/01/23 to 07/31/24
Outputs
Target Audience:Our target audience includes members of the scientific community focused on fish reproduction and physiology, aquaculturists, government risk assessors and policymakers, as well as biotechnology companies interested in genetic engineering technologies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training and growth opportunities for Mr. T. Umazume, who is currently pursuing a PhD, Mr. S. Samu, who holds an MS degree, and Mr. B. Tsai, a Bachelor of Science (BS) graduate. Each of them has made significant contributions to molecular and gene editing research, including the development of custom nucleases, embryo microinjection, and genotype and phenotype analysis. Additionally, two individuals received fish husbandry training through the project--one in a full-time position with a BS degree, and the other as a part-time undergraduate student. How have the results been disseminated to communities of interest? Presentation to the 9th International Workshop on the Biology of Fish Gametes in Leon, Spain. 9th International Workshop on the Biology of Fish Gametes 15th - 18th July 2024 León (Spain) with the Oral Communication "Partial inactivation of maternal effect gene to induce fish sterility" Sea grant aquaculture academic learning sessionVirtual Session #2, Monday, July 1, 2024: How and where genetic tools can be applied for responsible aquaculture production" Virtual presentation: Tuesday, May 21st,2024 Annual Project Director's Meeting: "Partial Inactivation Of Maternal Gene (PIMG) Technology For The Genetic Containment Of GE Fish" Presentation to the NRSP-8 Aquaculture group meeting in Raleigh, NC, 2023 "Precision Breeding to Control Gender and Fertility in Finfish, Optimize Production and Minimize aquaculture footprint" What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will outcross F1 nos3 3'UTR variantswith nos3mutants (nos3Δ5/+) to produce compound heterozygous (nos3Δ5/3'UTR-AR2,3,4). We will further incross nos3 3'UTR variants. To assay the maternal effect function of the 3'UTR mutations we will breed compound heterozygotes (dnd1Δ16/3'UTR-AR3, AR5, nos3Δ5/3'UTR-AR2,3,4) and F2 homozygous females (e.g.,nos33'UTR-AR2,3,4/3'UTR-AR2,3,4)with wild-type males, thenanalyze the PGCs in developing embryos. We expect that mutations in the cis-regulatory motifs will destabilize maternal RNA, leading toPGC ablation. This progeny will be raised to 3 months of age, at which point we will analyze the gonads at the morphological, cellular, and molecular levels to determine the extentof germline ablation.
Impacts
What was accomplished under these goals?
Objective 2: To increase the range of nos3 3'UTR regulatory motif disruption, we performed additional embryo microinjections using two new donor plasmids, pNos-AR2 and pNos-AR4, containing nucleotide modifications in two and four cis-acting stabilizing elements respectively. To assess integration of these variant 3'UTR sequences in F0 fish treated with any of five donor vectors (pNos-AR2, pNos-AR3, pNos-AR4, pDnd-AR3, pDND-AR5), we extracted genomic DNA from fins of fish with melanin pigmentation defects (around 10%) and conducted PCR of the target region and sequencing using Oxford Nanopore technology. Reads from 100 fish were aligned to the tilapia genome and analyzed with Crispresso software. We generated F0 tilapia mutants for the nos3 3'UTR, incorporating two, three, or four motif substitutions, and for the dnd1 3'UTR, with three or five motif substitutions, successfully confirming the integration of all donor sequences. In total, we established 20 founder lines, achieving an average germline 3'UTR replacement efficiency of 3%. Further analysis of mutation frequency in sperm and fin DNA from founder males revealed a positive correlation between mutation rates in somatic and germ cells, suggesting that the mutations were equally viable in both cell types. We found three F0 males (two dnd1 and one nos3 3'UTR variants) with a germline knock-in rates exceeding 90%, indicating that these mutations did not impair spermatogonia viability. Objective 3: We outcrossed the F0 dnd1 3'UTR founders with a tilapia line heterozygous for the loss-of-function dnd1 allele (dnd1Δ16/+). From this cross, we selected 163 F1 heterozygous fish (dnd13'UTR-AR3/+, dnd13'UTR-AR5/+) and 204 compound heterozygotes (dnd1Δ16/3'UTR-AR3, dnd1Δ16/3'UTR-AR5) representing the full spectrum of combinatorial mutations. F0 nos3 3'UTR founders were also outcrossed with wild-type (WT) lines, and the resulting heterozygous progeny (N=70) are being reared. These F1 fish were further divided into two groups of six mutants each for nos3 3'UTR and dnd1 3'UTR, considering the presence or absence of small deletions (scars) at the CrispR-Cas9 cut sites. Since dnd1 compound heterozygotes possess one loss-of-function allele and one intact coding sequence linked to a 3'UTR with altered cis-regulatory elements, the zygotic function of these mutated elements was assessed in the F1 generation. F1 females with up to five mutated cis-regulatory elements were dissected at 3-4 months (dnd1Δ16/3'UTR-AR3, dnd1Δ16/3'UTR-AR5), showing normal ovarian development. Males with dnd1Δ16/3'UTR-AR5 alleles produced functional sperm. These results suggest that the most extensive dnd1 3'UTR motifs alteration causes no harmful zygotic effects. In contrast, F1 compound heterozygous with a deleted 3'UTR region (dnd1Δ16/3'UTR-Δ170) were sterile, mimicking the dnd1 null mutant phenotype (dnd1Δ16/Δ16).
Publications
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Progress 08/01/22 to 07/31/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training and growth prospects for Mr. T. Umazume, holding a Master of Science (MS), Ms. Y. Takahashi with an MS degree, and Mr. S. Samu, also with an MS, along with Mr.T. Gilmore, who holds a Bachelor of Science (BS). Each of these individuals has made substantial contributions to the molecular and gene editing research, including the creation of customized nucleases, embryo microinjection, and the analysis of both genotypes and phenotypes. Additionally, two individuals received fish husbandry training under the project - one occupying a full-time position with a BS degree, while the other served as a part-time undergraduate student. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?To expand the pool of candidates with allelic replacement, we plan to conduct additional microinjections. Subsequently, we will rear the chosen fish to sexual maturity and cross the F0 males with wild-type (WT) females. The F1 offspring resulting from this cross will undergo PCR screening to identify individuals harboring the precise allelic replacement event. Upon identification, the male germ line mutants will be crossed with mutant lines carrying heterozygous null mutations in either nanos or dnd1 genes. This will enable the creation of individuals possessing only one functional copy of these genes, while being genetically linked to their modified 3'UTR.
Impacts
What was accomplished under these goals?
Objective 1: For further investigation of germ cell specific gene3'UTR regulatory mechanism, we selected fiveRNA stabilizing motifs in the dnd1 3'UTR and threein the nanos 3'UTR sequences. To ensure effective allelic replacement, we initially examined up to three pairs of gRNAs flankingthe 3'UTR genomic DNA regions of interest, comparing their editing efficiency and embryonic toxicity upon co-injectionwith Cas9 mRNA into fertilized eggs. Specifically, Cas9-treated embryos were injected with a gRNA targeting the tyrosinase gene, along with a set of upstream and downstream 3'UTR specific gRNAs. Across fivetreatment groups, survival rates varied, ranging from 1% to 19%. We then analyzed the frequency of indels at the upstream and downstream 3'UTR locations in theembryos displaying pigmentation abnormalities, resulting from successfull editingat thetyrosinase loci. Following a meticulous evaluation, we identified pairs that exhibited the highest editing efficiency with the least toxicity. We ultimately selected two combinations of gRNAs for removing the WT-dnd1 3'UTR sequence and one combination for excising the WT-nanos 3'UTR sequence. Objective 2: Building on the findings of objective 1, we opted to generate a total of four donor plasmids. The design of the nanos 3'UTR donor vector included nucleotide sequence modifications in three cis-acting stabilizing elements (pNos-AR3). For the dnd1 3'UTR donor vector, we integrated synthetic DNA with alterations in either three or five cis-acting stabilizing motifs (pDnd-AR3 and pDnd-AR5). To execute each allelic replacement experiment, we initially conducted a preliminary toxicity study assessing each design at three different donor DNA concentrations. One-cell stage tilapia embryos received an injection of a solution containing Cas9 mRNA, three sgRNAs, and the donor plasmid DNA at final concentrations of 12.5ng/ul, 20ng/ul, or 40ng/ul. Toxicity levels increased with rising DNA concentration, leading to survival rates ranging from 17% to 0.3%. For subsequent injections, we decided to use a final concentration of the donor vector set at 13ng, resulting in an average embryo survival rate of 10%. Approximately 10-30% of surviving embryos following injection exhibited a loss of the melanic pigment phenotype, indicating gene editing activity, with potential plasmid linearization and integration at the targeted loci. So far, from around 6000 embryos collectively injected using three donor vectors, we identified approximately 90 F0 candidate allelic replacement (AR) individuals exhibiting a mosaic pigment phenotype (N=29 for pDnd-AR5, N=58 for pNos-AR3, N=5 for pDnd-ARup). These candidates will undergo screening at sexual maturity to identify those displaying germ-line replacement of the 3'UTR sequences.
Publications
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