Abstract
BackgroundWith the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome.ResultsThe software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens.ConclusionsPhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1143-1) contains supplementary material, which is available to authorized users.
Highlights
With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable
Common designed Clustered regularly-interspaced short palindromic repeats (CRISPR) systems consist of expression of a Cas9 nuclease or nickase and a single guide Ribo nucleic acid (RNA)
This search is usually restricted for genomes of selected model organisms such as human, mice, fruit-fly, C. elegans, and yeast, making them irrelevant for researchers aiming to use CRISPR on other, less common, organisms
Summary
Several computational tools have been published aiming to identify candidates for CRISPR/CAS targeting. The third criteria compares the possibility to perform restriction site mutant screening analysis The statistics revealed that ~94 % and ~95 % of the genes in P. tricornutum and T. pseudonana, respectively, have atleast 1 potential guide RNA to be used as a Cas target against these genes Among the latter, most of the genes have high percent efficiency in terms of constituting mostly potential targets among total predicted Cas targets (Additional file 1: Figure S1). Out of the total, ~89 % genes in both the species (P. tricornutum and T. pseudonana) possess potential targets with restriction sites over the probable Cas cleavage site. Choosing such targets might help in fast screening of the mutants. The simple design of PhytoCRISP-Ex allows its use by end-users with moderate or no software programming background
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