Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) comprises of repetitive sequences that are separated by unique short DNA fragment acquired from a previously invading organism. In prokaryotes, the complex containing CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) and the CRISPR associated (Cas) protein could specifically target and cut the foreign DNA via base paring between the interspacer sequence and the target DNA. CRISPR/Cas has been developed as a new site-specific gene editing tool since 2012. In this system, a chimeric RNA and a single guide RNA (sgRNA) are expressed in a single molecule, which could be recognized by Cas9 protein. The specificity of CRISPR/Cas gene editing system is determined by the sequence of guide sgRNA that is complementary to the target region and usually contains the protospacer adjacent motif (PAM). Different Cas variants recognize different PAM, e.g. NGG for classic Cas9 protein, TTTV for most Cpf1 protein. Attracted by its simplicity, precision and highly efficiency, CRISPR/Cas has become a fundamental tool in various research areas including cereal crop breeding.
Wheat, maize and rice are the top three planting cereal crops worldwide. It is foreseeable that CRISPR/Cas gene editing system would bring great potential and now opportunities in precision breeding for cereal plants. To facilitate the application of CRISPR/Cas gene editing system in wheat, maize and rice, we developed a fast and precise sgRNA design tool, named CRISPR-Cereal for the above crops. Compared to other sgRNA design tools, CRISPR-Ceral has several advantages:
1. CRISPR-Cereal is fast enough to discovery the off-targets genome-wide with the maximal allowed mismatches up to 5 base pairs. In particular, it takes less than 49 seconds to scan for off-targets with 5 mismatches in wheat, which is a hexaploid with the genome size larger than 16 Gbp and contains about 85% repetitive elements2.
2. CRISPR-Cereal shows the putative restriction site at the -3 bp region proximal to PAM, and also gives information that whether the same restriction site existed in its 500 bp flanking region. This information allows the users to detect the mutation in a cost-efficient and straightforward manner.
3. CRISPR-Cereal performs off-target analysis to tell users whether the off-target sites located on the homologous genes from the A, B or D subgenome of wheat.
4. CRISPR-Cereal integrated the information of gene expression, chromatin openness, histones modification and DNA methylation of each candidate sgRNA, as the complex chromatin environment of the on-target sites in vivo would largely influence the binding activity of the Cas protein, and thus affect the editing efficiency.
5. CRISPR-Cereal considers the single nucleotide polymorphism (SNP) for each candidate sgRNA to assist sgRNA design in non-reference genome in breeding programme.
6. CRISPR-Cereal uses most updated or most widely used genome information for sgRNA design, especially the gap-free genome from O. sativa subsp. indica cv Minghui63 is included here.
1. He C, Liu H, Chen D, Xie WZ, Wang M, Li Y, Gong X, Yan W, Chen LL. CRISPR-Cereal: a guide RNA design tool integrating regulome and genomic variation for wheat, maize and rice. Plant Biotechnol J. 2021 Nov;19(11):2141-2143. doi: 10.1111/pbi.13675
2. Jiamin Sun, Hao Liu, Jianxiao Liu, Shikun Cheng, Yong Peng, Qinghua Zhang, Jianbing Yan, Hai-Jun Liu*, Ling-Ling Chen*. CRISPR-Local: a local single-guide RNA (sgRNA) design tool for non-reference plant genomes. Bioinformatics, bty970, https://doi.org/10.1093/bioinformatics/bty970
3. Hao Liu, Yuduan Ding, Yanqing Zhou, Wenqi Jin, Kabin Xie*, Ling-Ling Chen*. CRISPR-P 2.0: an improved CRISPR/Cas9 tool for genome editing in plants. Mol Plant, 2017, 10(3): 530-532. doi: 10.1016/j.molp.2017.01.003