- Open Access
PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing
© The Author(s). 2016
- Received: 15 January 2016
- Accepted: 22 June 2016
- Published: 1 July 2016
With 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.
The 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.
PhytoCRISP-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.
- Genome editing
Phytoplankton are microalgae that form an essential constituent of the marine food chain. Though microscopic and mostly uncharacterized, these minute organisms have tremendously showcased themselves as potential research models [22, 20]. Recent large-scale sampling to understand the morphological and genetic diversity of this hidden community [3, 12, 21] has already established the foundation for further molecular studies. The successful achievement of this exploration also reflects the interest of research communities towards the functional characterization of phytoplankton in the near future.
Clustered regularly interspaced short palindromic repeats CRISPR/CAS systems have recently emerged as a simple and accurate tool for genome editing , and show facile editing in numerous organisms including bacteria , yeast , plants , human  and other animals [2, 5, 7, 9, 17, 23, 24]. Common designed CRISPR systems consist of expression of a Cas9 nuclease or nickase and a single guide RNA (sgRNA). The latter includes a 20-bp target sequence used to target the Cas9/RNA complex to the desired chromosomal location. By modifying only these 20-bp, the targeting of the whole CRISPR/Cas9 complex will change and thus the DNA cleavage site. A well designed target sequence must not only optimally bind to its desired target, but must also not target any other site in the genome being edited, to avoid undesired off-target mutations . Several parameters were shown to contribute to a better targeting of the target sequence, and the general form of G-N19-NGG is widely accepted, although others were also suggested .
Because target site choice is a key point for promoting successful editing, several computational tools have been designed aiming to automate this procedure, all offering candidate target sites for a given input sequence/gene and potential off-target sites for a given background genome. 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. We designed PhytoCRISP-Ex, a user friendly web interface, as well as a stand-alone software, which predicts potential target sites for CRSIPR/CAS projects. The off-target analysis can be performed against indexed genomes from 13 algae (diatoms, green algae, haptophytes, etc.), or any user defined genome or transcriptome, assembled or not, making it useful for designing CRISPR projects for many communities.
There are two levels of filters and passing both designates the region as a potential sgRNA for Cas9 activity. The first level accepts the target sequence and tags it “PASS” if it has less than or equal to 2 base-pair mismatch with the closest off-target in the genome. The second filter, accepts the target if the seed region (last 15 bases including the PAM sequence) has 0 mismatch with off-targets anywhere in the genome. The pipeline reports the instances which are accepted by both or either one of the filters, but the ones which passes both the filters are designated as potential Cas9 targets.
Once the potential sgRNAs are filtered, they are then checked for the presence of none, one or more restriction sites corresponding to pre-selected and most common restriction enzymes (Additional file 1: Figure S1). Cas9 enzyme cleaves both DNA strands ~3–4 bases upstream of the PAM [19, 25]. Therefore along with the presence of restriction sites on the entire target sequence, PhytoCRISP-Ex reports specifically, if any, the restriction sites overlapping three and four bases upstream to the PAM sequence. Embedding a restriction site in the target sequence will help screening for mutants using PCR after a digest with the appropriate restriction enzyme. Presence and absence of the restriction sites along with its position on the sgRNA are reported in the output file (Fig. 1b). The list of restriction enzymes (Additional file 2: Table S1) can be updated and used when using the standalone version of the software. The PhytoCRISP-Ex pipeline is mounted with the index of 13 genomes and is accessible via web-server. An off-line standalone software is also developed which gives liberty to its users to use it for any genome, assembled or un-assembled. The standalone package also includes a few example files and a README file to help users install and execute PhytoCRISP-Ex. The standalone version can be found under “Download” tab at http://www.phytocrispex.biologie.ens.fr/CRISP-Ex/.
PhytoCRISP-Ex vs others
With the persuasive interest of scientific community towards phytoplankton research, the need of establishing genetic transformation tools for plankton species is thriving. PhytoCRISP-Ex provides a reliable and first ever application for predicting CRISPR/Cas9 targets within various plankton genomes. PhytoCRISP-Ex is also equipped with an easy to use, yet powerful, standalone version which gives its user the flexibility to use it on any genome. Many such and other unique features make the software more advance and appropriate for the use by a broad research community.
CAS, CRISPR associated protein; CRISPR, Clustered regularly-interspaced short palindromic repeats; DNA, Deoxyribo nucleic acid; PAM, Protospacer adjacent motif; PCR, Polymerase chain reaction; RNA, Ribo nucleic acid.
We thank Pierre Vincens for his help in mounting PhytoCRISP-Ex over the web-server. AR was supported by the Labex Memolife International doctoral program.
Availability of data and materials
Project name: PhytoCRISP-Ex
Project home page: http://www.phytocrispex.biologie.ens.fr/CRISP-Ex/
Compatible browsers: Mozilla, Internet Explorer, Chrome, etc.
Operating system(s): UNIX
Programming language: Shell, Perl, HTML
Any restriction to use by non-academics: None
AR, OM and LT conceived and designed the structure of the software. AR wrote the software. AR, OM CB and LT wrote the manuscript. LT coordinated the study. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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