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pubmed, cas, google, scholar, analysis, mutation, sgrna, piwil, tilapia, crisprcas, fragment, gene, ngs, rate, mutant, pgcs, nile, size, screening, methods, indel, editing, supplementary, phenotype, central, control, study, fig, mutants, larvae, data, cell, ngµl, nature, ads, number, zebrafish, total, article, germ, sgrnas, sequencing, high, melt, target, analysed, rates, genotypes, piwi, pcr,

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nature portfolio crispr-cas-derived rna-guided endonucleases privacy policy crispr/cas-mediated genome editing sgrna designing tools crispr/cas9-mediated genome editing nature 563 nature advertising performed research social media northern africa high-throughput genome editing vivo crispr/cas9-mediated hdr crispr/cas9-mediated provirus deletion crispr/cas9-mediated sterility studies reprints crispr/cas9 gene editing m13-tailed primers improve frame-shift mutation rates robust crispr/cas9 system rna-induced silencing complexes frame-shift mutation rate nuclease-specific cleavage products crispr/cas9-mediated efficient high gc-content applying crispr/cas9 methodologies microhomology-mediated end joining scientific procedures crispr-cas9 editing crispr/cas9-induced mutations crispr/cas9-mediated mutants multiplex genome engineering author correspondence original author zebrafish crispr editing screen crispr/cas9 efficacy permissions crispr/cas9-induced disruption paired-end reads belonged crispr nuclease system crispr/cas systems previously published results double-indexed library gene editing approaches reliable pcr-based method sybr green mix genome-wide binding determine editing efficiency genome engineering techniques

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         headline:Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system
         description:The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.
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