This application design CRISPR guide-RNAs (gRNAs) sequences for STAR Nucleases.
STAR 1.0 Nuclease gRNAs
A STAR 1.0 gRNAs is ~41 nt long, and it is composed by a constant
“scaffold” region (20nt, required for proper Art-STAR1.0 function) and a
variable “target” region (or “spacer”, 18-24 nt) that determines the
location in the genome that the nuclease will cut.
In order to make a cut at this location, the nuclease requires the
presence of a TTTN PAM (Protospacer Adjacent Motif) 5’ to the target
sequence in the genome, which serves as a “landing pad” for the enzyme
to bind and recognize the target region.
To increase its stability and efficacy, the 5’ and 3’ ends of the gRNA
can include chemical modifications.
STAR gRNAs
A STAR (Synthetic TrAceR guide RNAs) gRNAs is a complex of 2 shorter
RNAs: a “Modulator” and a “Targeter”.
When complexed with both the “Targeter” and “Modulator” of a STAR gRNA,
the STAR nuclease is able to cleave a target DNA similarly to regular
gRNAs.
The Modulator sequence is constant. The Targeter sequence is made by a
constant sequence followed by the variable spacer sequence.
Chemical modifications can be present at both 5’ and 3’ ends of both
Modulator and Targeter to improve the nuclease efficiency.
Art-STAR DNA cleavage
The STAR_1.0 nuclease DNA cleavage produces a “staggered” cut. The forward strand (the strand including the PAM and spacer sequence), is cleaved between the nucleotide 18-19 after the PAM, while the reverse strand is cleaved between the nucleotides 23-24. This creates “overhangs” at the ends of the cut DNA, which have complementary sequences to one another.
Design gRNAs modes
The App can design gRNAs from 3 input types:
- a DNA sequence
- genomic-coordinates
- a gene ID
Everytime the input paramenters change, the Update gRNAs button should be used to run a new gRNA design
Input DNA sequence
The Sequence Input tab allows the user to enter a DNA sequence to be targeted with one or more gRNAs. The app will then identify and return the spacer sequences that follow a compatible PAM in the input sequence. If the Search also minus strand option is selected, the app will also search for PAMs on the reverse complement of the input sequence. A table displays all of the gRNAs that are found, along with their respective start and end positions (relative to the input sequence), spacer sequence, and PAM.
Input genomic-coordinates
The Genomic positions input tab allows the user to enter the Chromosome name, start, and end coordinates of a target region in the Human genome (Hg38). The app will then extract the corresponding DNA sequence and display a list of gRNAs that have a cleavage site within the specified region. For each gRNA, the app will also show the PAM, spacer sequence, genomic coordinates, and cleavage site.
Input gene ID
The Gene ID Input tab allows the user to select a gene by
its ENSEMBL gene ID. The Find Ensembl ID tab can be used to
search gene IDs by gene name and or symbol.
The user select from a list of annotation types to be displayed
(i.e. whole gene, exons, CDS). By clicking on the desired annotation(s),
the user can specify the region they want to target with gRNAs. The app
will then extract the corresponding DNA sequence and display a list of
gRNAs with cleavage sites within the selected region. For each gRNA, the
PAM, spacer sequence, genomic coordinates, and cleavage site are
shown.
The Max distance of cleavage position from feature to consider
as overlap option (by default 0) can be increased to include
gRNA whose cleavage site is not immediately overlapping the target
region.
gRNA options
PAM
The compatible PAM sequences, indicated with IUPAC code.
N = any nucleotide
V = A, C or G
Y = C or T
CTTN PAMs are suboptimal for Art-STAR1.0
Recommended PAM: TTTV or TTTN.
Spacer length
Art-STAR1.0 can cleave target DNA with gRNAs with a minimal spacer
length of 18nt.
Recommended spacer length: 21nt.
Cleavage position
Art-STAR1.0 cleaves the target DNA between position 18 and 23 after the PAM. The cleavage position relative to the PAM (reported in the output) can be modified. This also affects the overlap between a gRNA cleavage site and a target region.
Download results
Click on “Download Table as CSV” to download the table of the gRNAs currently displayed.
WARNING:
The App output all gRNAs with a cleavage site overlapping the regions selected by the user, but does not check whether the gRNAs can target also other sites in the genome.
After designing gRNAs targeting the desired regions, the user must check their specificity using a dedicated tool (e.g CatsOFFinder)