|A Fast and Comprehensive Guide RNA Design Tool for Genome Editing, Repression and Activation.|
Four manipulations are allowed in CRISPR-ERA. For gene editing using nickase, a pair of sgRNA is found in gene editing region of target gene within a offset distance (-50 bp, +100 bp). For gene editing using nuclease, sgRNA is found in exon region of target gene. Targeted exon is given in final sgRNA design result. For gene repression, sgRNA is found within -1500bp upstream TSS to 1500bp downstream TSS region of target gene. For gene activation, sgRNA is found within -1500bp upstream TSS to TSS region of target gene.
The databases used for different organisms.
Organisms allowed under different manipulations
Input gene name
Official symbols are allowed, other kind of gene symbol can’t be recognized by CRISPR-ERA. For example, for gene Pou5f1(Oct4) http://www.ncbi.nlm.nih.gov/gene/5460 in human, Oct4 can’t be found, then result webpage outputs “Gene name cannot be found. Please input alternative symbols”.
For gene editing using nickase and nuclease, the location should be a DNA region for targeting, such as chr1:4000000-4001000, CIRSPR-ERA searches for all possible sgRNAs in this region. For gene repression and activation, the TSS location for the target gene should be a site in chromosome, such as chr1:4000000, and the strand should be given. CRISPR-ERA searches for sgRNA in the region (-1500bp, +1500bp), (-1500bp, 0) relative to TSS for gene activation and gene repression, respectively.
Input gene sequence
Sequence can be given in fasta format or not. Users can input the sequence in the textbox or upload locally. Sequence length must be shorter than 5kbp, or users can find the sequence location in UCSC genome browser and choose “Location” in step3.
When clicking on the E score and S score columns, the detail will be presented.
E score detail:
GC content and Polyt information. GC content：0.8 the GC content of the sgRNA is 0.8.
Polyt: No there is no TTTT in sgRNA.
S score detail:
For gene editing using nuclease, gene activation and gene repression, S score detail has two parts: PAM= NGG within 2 mismatches, and PAM =NAG within 2 mismatches. If the total sgRNA number is less than 6, the location of each offtarget will be presented.
For example :
PAM=NGG:[0mismatch,1mismatch,2mismatches]=[0,0,0] [.,.,.] PAM=NAG:[0mismatch,1mismatch,2mismatches]=[0,1,0] [.,chr11:123572734,.]
(For this sgRNA, there is only one offtarget with 1 mismatch and an NAG PAM sequence. The location is chr11:123572734 ).
(PAM=NGG)[0mismatch,1mismatch,2mismatches]=[1,2,0] [chr12:8287137,chr8:128428398;chr1:155403258,.] (PAM=NAG)[0mismatch,1mismatch,2mismatches]=[0,0,0] [.,.,.]
(For this sgRNA, there are 3 offtargets when PAM sequence is NGG. One with 0 mismatch which binds to chr12:8287137. Two with 1 mismatch which bind to chr8:128428398 and chr1:155403258).
For gene editing using nickase, offtarget of sgRNA pair will be presented.
For example .,. no offtarget is found within 2 mismatches.
There are two offtargets, one sgRNA offtarget pair both have 0 mismatch which bind to chr14:74036555 and chr14:74036565, respectively. The other sgRNA offtarget pair has 2 mismatch with sgRNA1 and 1 mismatch with sgRNA2, which bind to chr14:74059088 and chr14:74059098, respectively.
CRISPR-ERA computes efficacy score (E) and specificity penalty score (S) for each sgRNA according to off-target, location, GC content, poly-T sequence, etc. Both E and S start with 0, and accumulate with the criteria listed in tables below.
Efficacy score computation rules for non-bacteria
Efficacy score computation rules for bacteria.
Off-target number for sgRNA pair is OFF(ma,mb), where ma, and ma are the number of mismatches of the off-target for sgRNAa and sgRNAb in the pair, respectively.
Specificity score for gene editing using nickase. Off-target number for sgRNA pair is OFF(a,b), where a, b are the mismatches of the off-target for each sgRNA in the pair, respectively.
In the result webpage, the score E+S is given, which is the sum of specificity score and efficacy score, and it will be presented in different color in UCSC genome browser
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