gRNA Designer for Zebrafish

You can submit a refseq gene ID and exon number (also nucleosome organizaiton and chromatin accessibility status, optional) to get potential Cas9 cleavage target sites with following information:

1. Genomic location

2. Nucleosome organizaiton status

3. Chromatin Accessibility status

4. Sequence feature evaluation

5. Potential off-target site evaluation

Gene name query

Your gene:

Exon number:


Nuclesome organization status:



Chromatin accessibility status:







In this websever, we scanned all potential CRISPR/Cas9 targets on zebrafish genome exons (Refseq annotation, with NGG as PAM sequence) and evaluated each targets from the following four aspects:

1. Nucleosome organizaiton status labeled by "NL", "NO" or "Dynamic"

2. Chromatin Accessibility status labeled by "OC" or "CC"

3. Sequence feature evaluation calculated by SSC

4. Potential off-target site evaluated by sequence similarity with other site of genome

Once an user submitting a query via a gene name (both Refseq ID and official gene symbol are supported), the matching records on NL/OC region and with few potential off-target site will be given high priority to return to users. If no preferential targets returned, all matching result will be returned.

Briefly, the four aspects of evaluation standard were identified as follow:

1. Nucleosome Organization Status Identification
  • MNase-seq data of the 256-cell and dome stage from our previous study were involved (GSE44269). To identify the nucleosome midpoints, 73 bp was added to the 5’ end of the coordinate for each mapped read. Then, all nucleosome midpoints in two stages were combined to predict stable nucleosomes using GeneTrack (with parameters -e 147 -s 20). Among all nucleosome peaks predicted by GeneTrack, we removed those with low enrichment scores and merged the remaining peaks, with those presenting lengths shorter than 180 bp identified as nucleosome occupied regions. The other merged peaks were regarded as dynamic nucleosome regions within the 256-cell and dome stage. Subsequently, the complementary regions of merged peaks longer than 10 bp and shorter than 100 bp were identified as nucleosome linker regions. In summary, zebrafish genome region was divided into nucleosome linker (NL) regions, nucleosome occupied (NO) regions and nucleosome dynamic regions.
  • We regard gRNAs targeting on NL region as potential high efficiency gRNAs.
    • 2. Chromatin Accessibility Status Identification
  • The ATAC-seq reads of 256-cell and dome stages were aligned to the zebrafish genome built using zv9/danRer7 using bowtie2 (v 2.2.3). We removed the reads that were mapped to the scaffold chromosome and the reads with MAPQ lower than 30. The remaining reads were used to call peaks using the MACS2 (v 2.0.10.20131028) (callpeak function with parameter -q 0.01 –SPMR. We combined the peaks called from the two stages. Each peak summit was extended 1000 bp upstream and 1000 bp downstream, and the merged regions were identified as open chromatin (OC) regions. The remaining regions on the genome were regarded as close chromatin regions (CC) regions.
  • We regard gRNAs targeting on OC region as potential high efficiency gRNAs.
    • 3. Sequence Feature Score Calculation
  • For sequence feature evaluation, we used the source code of Sequence Scan for CRISPR (SSC source code) which includes two core command Fasta2Spacer and SSC. Firstly, we used bedtools getfasta function to generate 40bp sequence for each target. Secondly, Fasta2Spacer command was used to scan each fasta record to get target site. Then SSC scores for each targets were calculated by SSC command.
  • We regard gRNAs with higher SSC score as potential high efficiency gRNAs.
    • 4. Potential Off-target Sites Counting
  • For each potential target, we evaluated the possibility of off-target binding by calculating 0-2 mismatched sites on the zebrafish genome. We used bowtie version 1.0.1 to output all 0-2 mismatched sites on the genome with -v 2 -a parameters, and then a custom Python code was used to calculate the mismatched site number for each target. We regard targets with more similar site on genome as high-possibility-off-target sites.
  • The result format 'x:y:z' represents for each gRNA targets, there are x other sites in zebrafish genome which are 0-mismatch similar with target site, y sites for 1-mismach and z sites for 2-mismatch. Generally, CRISPR/Cas9 potential off-target sites tend to locate at similar-sequence sites on genome. Therefore, we recommand users to design gRNA on the targets with less similar sequence site.