6 research outputs found
Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9- mediated HDR in Atlantic salmon
Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.publishedVersio
Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon
Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.publishedVersio
Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9- mediated HDR in Atlantic salmon
Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general
Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors
New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon
Risk of recurrence in resected IPMN: Preliminary results from a multi-institutional international study
European evidence-based guidelines on pancreatic cystic neoplasms
Evidence-based guidelines on the management of pancreatic cystic
neoplasms (PCN) are lacking. This guideline is a joint initiative of the
European Study Group on Cystic Tumours of the Pancreas, United European
Gastroenterology, European Pancreatic Club, European-African
Hepato-Pancreato-Biliary Association, European Digestive Surgery, and
the European Society of Gastrointestinal Endoscopy. It replaces the 2013
European consensus statement guidelines on PCN. European and
non-European experts performed systematic reviews and used GRADE
methodology to answer relevant clinical questions on nine topics
(biomarkers, radiology, endoscopy, intraductal papillary mucinous
neoplasm (IPMN), mucinous cystic neoplasm (MCN), serous cystic neoplasm,
rare cysts, (neo)adjuvant treatment, and pathology). Recommendations
include conservative management, relative and absolute indications for
surgery. A conservative approach is recommended for asymptomatic MCN and
IPMN measuring < 40 mm without an enhancing nodule. Relative indications
for surgery in IPMN include a main pancreatic duct (MPD) diameter
between 5 and 9.9 mm or a cyst diameter >= 40 mm. Absolute indications
for surgery in IPMN, due to the high-risk of malignant transformation,
include jaundice, an enhancing mural nodule > 5 mm, and MPD diameter >
10 mm. Lifelong follow-up of IPMN is recommended in patients who are fit
for surgery. The European evidence-based guidelines on PCN aim to
improve the diagnosis and management of PCN