CRISPR
Donor Design
The CRISPR Donor Design tool can be accessed by clicking the
"CRISPR Donor Design" link under CRISPR tools section. User needs
to login to the application to access it.
Introduction
Crispr single strand short oligo design for crispr in vivo
genome-editing. Crispr gRNA could be located at exon region,
intron region or across intron/exon boundary region.
1. When gRNA is located intron region, our ssODN consider
following three factors:
* Allowing users to make point mutation, multi-base replacement, insertion and deletion.
* Synonymous mutation at PAM sequence and their codon usage optimization in different species,
* If no silent mutation at PAM found, synonymous mutations at nucleotide close to PAM are created
* new restriction enzymes introduced by new mutations created by users
2. When gRNA is located intron region
* Allowing users to make point mutation, multi-base replacement, insertion and deletion.
* Mutations at PAM sequence ,
* new restriction enzymes introduced by new mutations created by users
3 When gRNA is located across intron/exons
* Allowing users to make point mutation, multi-base replacement, insertion and deletion.
* check whether PAM is at exon seq or not, if in exon, create silent mutation as described in 1. otherwise, design gRNA silent mutations
following the procedure as described in 2),
* new restriction enzymes introduced by new mutations created by users
In our design page, we list all possible ssODNs, users can
choose the ones to save in the design result database.
File
Preparation
1. In order to do ssODN design, users first need to choose
sequence files, the sequence files can be chosen from file that
was saved in the database
2. Users can load file from local or retrieve from NCBI data
base
3. Load file from local by clicking on "Load File"
4. Retrieve sequence from NCBI by clicking in "Retrieve
Sequence From NCBI" and choosing genome and gene symbol/id
User can continue designing ssODN from this page or he can go back
to the library page and continue by clicking on "ssODN
Design".
ssODN design
User need chose protein/transcript id, gRNA sequence that
bear PAM seq, define donor size (default 120 nt), and donor name
that will be used to save to database
Visualization
Gene structure visualization and sequence pane for ssODN
design
Mutation
Make mutation at the sequence close to gRNA by selecting
base(s).
Point
Mutation, Deletion or Substitution
For a selected single or muti-nucleotide, user can create
substitution mutation, or make a deletion by:
* Use mouse select dna base (labelled with yellow color)
* Then click left key
* Sub-menu is poped out
Select any of the options for mutation, a separate pop-up
window let the user design ssODNs
Substitution
Point Mutation(SNP) User can point mutate by
selecting an alternate for the selected base from the options
provided
when it is done, the page will be directed to design page to
show ssODNs
Insertion
For a selected position, user can make insertion
On clicking the "Insert Bases" option, a separate
pop-up window let the user add base(s) to be inserted
when it is done, the page will be directed to design page to
show ssODNs
Donor
Design Result
After the mutation, a separate pop-up window shows the
result with all the ssODN(s)
If there are multi-ssODN,
* Click the check box against the ssODN you need
* Click "Save" (if no checkbox is selected the first ssODN will be saved as default)
Saved Designs
All the saved ssODN(s) will be saved under "Saved Designs"
tab
Download
Saved Designs tab help user download the saved results in
the form of pdf file.
This is a downloaded design file
