https://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1253&context=faculty_pubs

RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals

A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods.

Genetic Inhibition by Double-Stranded RNA

Short interfering RNAs (siRNAs) are double-stranded RNAs of ’21‐25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates siRNAs have a characteristic structure, with 5*-phosphatey3*-hydroxyl ends and a 2-base 3* overhang on each strand of the duplex In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells

/pdf/specific-inhibition-of-gene-expression-by-small-double-4ot4xfxzyk.pdf

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

/pdf/targeted-mrna-degradation-by-double-stranded-rna-in-vitro-4rjezufkc9.pdf

Targeted mRNA degradation by double-stranded RNA in vitro

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

Rna interference mediating small rna molecules

Double-stranded RNA induces specific developmental defects in zebrafish embryos.

Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.

/pdf/dnd-knockout-ablates-germ-cells-and-demonstrates-germ-cell-4jt5010wt8.pdf

Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

Wild and domesticated Atlantic salmon males display large variation for sea age at sexual maturation, which varies between 1–5 years. Previous studies have uncovered a genetic predisposition for variation of age at maturity with moderate heritability, thus suggesting a polygenic or complex nature of this trait. The aim of this study was to identify associated genetic loci, genes and ultimately specific sequence variants conferring sea age at maturity in salmon. We performed a genome wide association study (GWAS) using a pool sequencing approach (20 individuals per river and phenotype) of male salmon returning to rivers as sexually mature either after one sea winter (2009) or three sea winters (2011) in six rivers in Norway. The study revealed one major selective sweep, which covered 76 significant SNPs in which 74 were found in a 370 kb region of chromosome 25. Genotyping other smolt year classes of wild and domesticated salmon confirmed this finding. Genotyping domesticated fish narrowed the haplotype region to four SNPs covering 2386 bp, containing the vgll3 gene, including two missense mutations explaining 33–36% phenotypic variation. A single locus was found to have a highly significant role in governing sea age at maturation in this species. The SNPs identified may be both used as markers to guide breeding for late maturity in salmon aquaculture and in monitoring programs of wild salmon. Interestingly, a SNP in proximity of the VGLL3 gene in humans (Homo sapiens), has previously been linked to age at puberty suggesting a conserved mechanism for timing of puberty in vertebrates.

/pdf/the-vgll3-locus-controls-age-at-maturity-in-wild-and-3m5jjaqeox.pdf

The vgll3 Locus Controls Age at Maturity in Wild and Domesticated Atlantic Salmon (Salmo salar L.) Males

Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.

/pdf/targeted-mutagenesis-in-atlantic-salmon-salmo-salar-l-using-2xfvdoa36i.pdf

Anna Wargelius

Papers

Double-stranded RNA induces specific developmental defects in zebrafish embryos.

Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

The vgll3 Locus Controls Age at Maturity in Wild and Domesticated Atlantic Salmon (Salmo salar L.) Males

Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation.

Potential of Genome Editing to Improve Aquaculture Breeding and Production