CRISPR-offinder Usage

Overview

CRISPR-offinder provides a simple and easy way to design optimal sgRNAs for user defined protospacer adjacent motif (PAM).

Input your DNA sequence (format fasta, sequence length < 500bp)

Users can easily design sgRNAs by paste or uploaded FASTA sequence as below, and may not exceed 500 bp.

Length of protospacer - Allowed range (nt) of protospacer length of the candidate sites.

PAM requirement

1. sequence requirement :

  • NGG - SpCas9 from Streptococcus pyogenes - direction: 3’
  • NRG - SpCas9 from Streptococcus pyogenes - direction: 3’
  • NNAGAAW - StCas9 from Streptococcus thermophilus - direction: 3’
  • NNNNGMTT - NmCas9 from Neisseria meningitidis - direction: 3’
  • NNGRRT - SaCas9 from Staphylococcus aureus - direction: 3’
  • NNNRRT - SaCas9 KKH variant - direction: 3’
  • NGG(reduced NAG binding) - SpCas9 D1135E variant - direction: 3’
  • NGCG - SpCas9 VRER variant - direction: 3’
  • NGAG - SpCas9 EQR variant - direction: 3’
  • NGAN-NGNG - SpCas9 VQR variant - direction: 3’
  • NGG - FnCas9 from Francisella novicida - direction: 3’
  • YG - FnCas9 RHA variant - direction: 3’
  • TTTN - AsCpf1 from Acidaminococcus, LbCpf1 from Lachnospiraceae - direction: 5’
  • TTN - FnCpf1 from Francisella novicida strain U112 - direction: 5’
  • CTA - FnCpf1 from Francisella novicida strain U112 - direction: 5’
  • TTN-CTA - FnCpf1 from Francisella novicida strain U112 - direction: 5’
  • TTN - C2c1 from four major taxa: Bacilli, Verrucomicrobia, a-proteobacteria, and d-proteobacteria - direction: 5’
  • Custom, Enter your PAM - enter user defined-PAM

Note that CRISPR-offinder allows mixed bases to account for the degeneracy in PAM sequences

Code Base Code Base
A Adenine K G or T
C Cytosine M A or C
G Guanine B C or G or T
T Thymine D A or G or T
R A or G H A or C or T
Y C or T V A or C or G
S G or C N any base
W A or T    

2. direction requirement - 3' or 5'

3. location requirement

  • sense strand - PAM located on the sense strand
  • anti-sense strand - PAM located on the anti-sense strand
  • both strands - PAM located on the both DNA strands

Note that if user choose b(both strands), the hidden buttons of "Single-gRNA" and "Paired-gRNA" will be shown, if user choose "Paired-gRNA", another hidden buttons of "gRNA offset" will be shown, user can set the value of (minimum) min and (maximum) max for designing Paired-gRNAs.

Parameter setting

GC% - GC contents of protospacer

Mismatches Number - The maximum number of mismatches that allowed in the "gRNA" region when perform whole genome alignment, 'N' in PAM sequence are not counted as mismatched bases.

Specificity check (target genome)

Or paste your genome sequence (format fasta, sequence length < 10kb)

Note that small genome sequence which from bacteria or virus which less than 10 kb was allowed to paste directly.

Result page

An example of output file for designing of single sgRNA

  1. Once the user clicks on this button, the print dialog of the browser will open.
  2. Once the user clicks on this button, the download dialog of the browser will open.
  3. Once the user clicks on this button, the result in the table will rank.
  4. Right click on selected column with the mouse, the hidden dialog of the browser will open, user can choose to print, download or view off-target sites.

An example of output file for viewing off-target sites

  • First column - given query sequence
  • Second column - FASTA sequence title (if you downloaded it from UCSC or Ensembl, it is usually a chromosome name)
  • Third column - position of the potential off-target site
  • Forth column - actual sequence located at the position (mismatched bases noted in lowercase letters)
  • Fifth column - indicates forward strand(+) or reverse strand(-) of the found sequence
  • Last column - the number of the mismatched bases ('N' in PAM sequence are not counted as mismatched bases)

An example of downloaded result for Paired-gRNAs

  • First column - sgRNA ID
  • Second column - Target sequence, which located on the anti-sense strand
  • Third column - start position of the sgRNA target site in given query sequence
  • Forth column - end position of the sgRNA target site in given query sequence
  • Fifth column - GC contents of protospacer
  • Sixth column - sgRNA ID
  • Seventh column - Target sequence, which located on the sense strand
  • Eighth column - start position of the sgRNA target site in given query sequence
  • Ninth column - end position of the sgRNA target site in given query sequence
  • Tenth column - GC contents of protospacer
  • Last column - sgRNA offset for paired-gRNA