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.
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
||G or T
||A or C
||C or G or T
||A or G or T
||A or G
||A or C or T
||C or T
||A or C or G
||G or C
||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.
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.