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Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

Gandhi, Shashank and Christiaen, Lionel and Stolfi, Alberto (2016) Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona. . (Submitted) https://resolver.caltech.edu/CaltechAUTHORS:20160516-094256455

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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.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1101/041632DOIDiscussion Paper
ORCID:
AuthorORCID
Gandhi, Shashank0000-0002-4081-4338
Christiaen, Lionel0000-0001-5930-5667
Additional Information:The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint first posted online Feb. 28, 2016. This article is a preprint and has not been peer-reviewed. We are grateful to Florian Razy-Krajka, Farhana Salek, and Aakarsha Pandey for discussions and technical assistance; Tara Rock for advice on next-generation sequencing; Rahul Satija for sequencing the libraries and for his invaluable insights into the sgRNA sequence analysis; Shyam Saladi, Elena K. Perry, and the High Performance Computing team at NYU for their help troubleshooting the bioinformatic analysis. This work was funded by the NIH R01 GM096032 award to L.C., an NYU Biology Masters Research Grant to S.G., while A.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology [NSF-1161835].
Funders:
Funding AgencyGrant Number
NIHR01 GM096032
New York University (NYU)UNSPECIFIED
NSF Postdoctoral FellowshipNSF-1161835
Record Number:CaltechAUTHORS:20160516-094256455
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160516-094256455
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67111
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:16 May 2016 19:47
Last Modified:23 Oct 2020 18:10

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