Help Index

1. Flowchart of gRNA Design

2. Comparison with Other Tools

3. Input Preparation

4. Parameters Introduction
     4.1 Basic Options
     4.2 Advanced Options

5. Result Interpretation
     5.1 Running Information
     5.2a Result Table for pgRNA
     5.2b Result Table for sgRNA Pair

6. Search in Database

1. Flowchart of gRNA Design flowchart.png

2. Comparison with Other Tools

Input: GuideScan permits genomic region (chr1:38140090-38140189 in hg38), and the others take target sequence as input.
Target sequence:

Comparison Result:
1) SSC designs 30bp sgRNA for scoring, thus missing the last sgRNA would appear in 20bp sgRNAs.
2) WGE outputs 28 lines. Actually 22 sgRNAs were remained after deleting the repeated sgRNAs.
3) OT: Off-targets.
4) GuideScan only provides the off-target information when mismatch is equal to 2 or 3.

3. Input Preparation

pgRNAFinder accepts input in three formats by typing or uploading:

1) Genomic region (chr:start-end)
2) Gene name
3) Target sequence (fasta)


By clicking “genomic region”, “gene name” or “target sequence”, a corresponding example will be shown in the text box, which can be used to test this web tool.

4. Parameters Introduction

4.1 Basic Options


gRNA Type: Paired Nickase, Paired gRNA, Single gRNA.
1) Genome: hg19, hg38, mm10, bosTau, canFam, danRer, galGal, ratNor, sacCer or susScr.
2) PAM sequence: NGG, NAG, NNGRRT, NNNNACA or self-definded sequence comprised of A,G,C,T,N,R.
3) On-Target: Exon or All
     Exon: At least one gRNA whose on-target site locates in exon region (recommended).
     All: Report all gRNAs.
4) SSC score cutoff: For evaluating gRNA efficiency, higher is better, 0.5 is recommended; -1~1 (may be a little lower than -1 or higher than 1).
5) Mismatch: Mismatch for Off-Spotter when finding off-targets.
6) Max Off-Targets: Off-targets located in genome.
7) Max OTs in Exon: Off-targets located in exon.

The following options are available only when "gRNA Type" is set to "Paired".
8) Strand: Same (two gRNAs are in the same stand), Diff (two gRNAs are in the different stand), Both.
9) Pair Distance: Distance between paired gRNA.
10) Coverage: Equal to distance between pgRNA divided by the length of sequence.

4.2 Advanced Options


Advanced options are hidden by default. Choose "Show" to show them.
1) Restriction Enzymes:No or GAGACC or GAGACC,GGTCTC or self-defined like e1,e2,...en. Guide RNAs containing with restriction enzyme site will be filtered out.
2) gRNA Length: The number of base in the upstream of PAM; at better >=20bp for off-target analysis.
3) Downstream Length: The number of base in the downstream of PAM.
4) CG Content.

After setting up the options, please click Submit to run the tool.

5. Result Interpretation

5.1 Running Information

As below. Step 5 is the most time consuming, which would take from minutes to hours depending on the workload, please wait patiently especially after you submit a lot of work. The hyperlink to your results will be sent to your mailbox.


5.2a Result Table for pgRNA

The result are provided in several different ways. You can also choose the columns you are interested to view, but it will not affect the file you export.


The explanation to each column:

Column NameMeaning
query_infoName of input sequence
gRNA1Base sequence of one gRNA
gRNA2Base sequence of another pairwise gRNA
pos1Position of gRNA1 in input sequence
pos2Position of gRNA2 in input sequence
strand1Strand of gRNA1
strand2Strand of gRNA2
SSC_score1Efficiency score for gRNA1 by SSC software
SSC_score2Efficiency score for gRNA2 by SSC software
on_target1On-target site of gRNA1
on_target2On-target site of gRNA2
on_target_pairOn-target site of knockout region between pgRNA
phastCE1Phast conserved element overlapping with gRNA1
phastCE2Phast conserved element overlapping with gRNA2
phastCE_pairPhast conserved element overlapping with knockout region between pgRNA
#ot1_in_exonThe number of off-targets located in exon for gRNA1
#ot2_in_exonThe number of off-targets located in exon for gRNA2
#ot_pair_in_exonThe number of off-targets located in exon for the pgRNA
#ot1The number of off-targets for gRNA1
#ot2The number of off-targets for gRNA2
#ot_pairThe number of off-targets for the pgRNA
CG1CG content of gRNA1
CG2CG content of gRNA2
pair_disDistance between gRNA pair
coverCoverage of gRNA pair
del_freqDeletion frequency, this value depends on the distance between pgRNA
ot_site1Detailed off-target sites for gRNA1
ot_site2Detailed off-target sites for gRNA2

More detailed explanations can be found in README.txt.

5.2b Result Table for sgRNA


The explanations are consistent with the "pgRNA" above.

6. Search in Database (Reserved)