CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants

Abstract

Precise and efficient genome editing is very important for gene functional characterization. In recent years, sequence-specific DNA nucleases have been developed to increase the efficiency of gene targeting or genome editing in animals and plants. Among them, Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are two most commonly used sequencespecific chimeric proteins (Gaj et al., 2013). Recently, a breakthrough gene-targeting tool based on RNA-guided Cas9 nuclease from type II prokaryotic Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated system has been developed (Jinek et al., 2012). CRISPR/Cas system is an adaptive defense system in prokaryotes to fight against alien nucleic acids (Horvath and Barrangou, 2010). The CRISPR loci are variable short spacers separated by short repeats, which are transcribed into synthetic single-guide RNA (sgRNA). The sgRNA forms a functional complex with CRISPR-associated nuclease (Cas9) and guide the nuclease to genomic loci matching a 20-bp complementary invading DNA, cleaving it immediately upstream of a required 5'-NGG Protospacer Adjacent Motif (PAM). A chimeric sgRNA that mimics the natural synthetic sgRNA can be used to target Cas9 for genome editing in eukaryotic cells.Until now, the CRISPR/Cas system has been successfully applied to efficient genome editing in bacteria, animals and plants (Jiang et al., 2013; Mali et al., 2013; Xie and Yang, 2013). To inform the selection of target sites and avoid off-target effects, Hsu and colleagues characterized Cas9 targeting specificity in human cells by a volume of experiments, and built a computational tool for optimized sgRNA selection in human and animal genomes (Hsu et al., 2013). However, with the wide application of CRISPR-system in plant genome editing, tools for optimized sgRNA selection in plants are highly needed. In the current analysis, we present a web application tool-CRISPR-P, for CRISPR sgRNA design in more than 20 plant species. CRISPR-P allows users to search for highly specific Cas9 target sites within DNA sequences of interest, which also provides off-target loci prediction for further analysis and marks restriction enzyme cutting sites for convenience. CRISPR-P is freely available at http://cbi.hzau.edu.cn/crispr/.Genomic data and annotation of the analyzed plant genomes are obtained from public databases. The genome and annotation of Arabidopsis lyrata (v.1.0), Arabidopsis thaliana (TAIR10), Brachypodium distachyon (v1.0), Brassica

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response

“…The hda19-5 and hda19-6 alleles were generated from the same sgRNA. The sgRNAs used for mutagenesis in the generation of them were designed using the CRISPR-P program (Lei et al, 2014).…”

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

“…A similar candidate target was suggested by three CRISPR designing tools (CRISPOR, CRISPR-P, and CRISPR-PLANT; Lei et al, 2014;Xie et al, 2014;Haeussler et al, 2016) and was selected for mutagenesis. A protospacer was designed using the ATTG_protospacer and AAAC_protospacer primers (Supplemental Table 3), and the annealed primers were cloned into pEn-Chimera according to Fauser et al (2014).…”

Exploiting genome variation to improve next-generation sequencing data analysis and genome editing efficiency in Populus tremula × alba 717-1B4

“…Custom Perl scripts were used for probe annotation following the NetAffx procedures (Liu et al 2003). gRNAs were designed according to the method of Lei et al (2014) for 1000 randomly selected genes extracted from the P. trichocarpa or sPta717 genome. The resulting gRNA sequences were mapped to the respective genome using BatMis (Tennakoon et al 2012) to evaluate specificity according to Hsu et al (2013).…”