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• W3100447102 startingPage "041632" @default.
• W3100447102 abstract "The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a CRISPR/Cas9-induced Ciona Knock-Out (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona." @default.
• W3100447102 created "2020-11-23" @default.
• W3100447102 creator A5014613784 @default.
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• W3100447102 date "2016-02-28" @default.
• W3100447102 modified "2023-09-28" @default.
• W3100447102 title "Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona" @default.
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• W3100447102 hasPublicationYear "2016" @default.
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