getCRISPRguideRNA evaluation tool for CRISPR design
getCRISPR is a custom-built web resource for the design of guide RNAs for editing in plants. This tool contains mainly customizations that you won’t find in other guide RNA design tools.
Select species and genome DB
The selected genome db will be used for target sequence,primer pairs and restriction enzyme search if you use the “specify a known genomic region” section. And also, all the chromosomes on this genome db will be used for off-target search for both “specify a known genomic region” and “specify your own sequence” sections.
Specify a genomic region
You can enter the name of a single gene or genomic position to search target sequences, primer pairs and restriction enzymes. You can also enter multiple genes to see the best target sequence for each of them. You can click on a target sequence to see the off-targets, primer pairs and restriction enzymes for this target sequence. The offtargets will be searched on all the chromosomes in genome db selection in the dropdown. The gene names, chromosome ids and coordinates should match the gene names, chromosome ids and coordinates of genome db that you chose in dropdown.
Alternatively, you can enter your own sequence to make the target sequence,primer pairs and restriction enzyme search on this sequence. The offtargets will be searched on all the chromosomes in genome db selection in the dropdown.
Select gene region settings
These settings are valid for only if you selected single or multiple gene options above. You can select “Coding region” to search on all exons except for 5 UTR and 3 UTR regions. You can select “Splice sites” to see the all the targets start in an exon and end in an adjacent intron or vice versa. You can also select “All exons”, “5 UTR” and “3 UTR” options or you can type in “Only target exon(s)” textbox more than one exon id that you target as a search region.
Select gRNA settings
The target sequences will have the PAM(Protospacer Adjacent Motif) sequence you select here at the end. You can select “GN or NG” or “GG” options if you want to have targets beginning with “GN or NG” or “GG” on 5' side of targets. Also, you can select “No requirements” for targets that can begin with any nucleotide.
5′ requirements for gRNA
gRNA length (without PAM)
Select off-target settings
You can select off-target search method and click the links for papers for each method.
Select primer settings
Product size is the distance(number of nucleotides) between left and right primers. Primer size is number of nucleotides in primer. Primer Tm(Primer Melting Temperature) is the input paramater for Primer3 program which is used here for finding primer pairs. You can also set a minimum distance from primer to target sequence.
You can choose your preference for company and minimum length of recognition site for restriction enzymes. You can see them by clicking on the target sequence at the result page.