My goals are to use a free webtool to:
- Identify guide RNAs (direct-repeat sequence followed by the targeting sequence) appropriate for use with AsCpf1 in order to target a specific segment of genomic DNA.
- Estimate efficiency and specificity of using the guide RNAs identified by the tool.
Unfortunately, when I tried to do this using the CRISPOR tool, the "Job Status" never advanced past the "Waiting" stage, even after 10 minutes. Attached is a screenshot to show what I see.
Specifically, I used the following settings in my query:
I named my sequence:
Mm Acaca block2 from BLAT alignment
In "Step 1" I submitted this sequence:
aactaaatct ccagcatctc catccccttc ttaggtttat ttattttatg ggtatgagtg
tatgtccgtg caccacatgt gtgcctggtg tctaccaagg taagtagagg tatacaaacc
cttggaattg aattatccac catgccgtca ggtgctggga gcaaattcag gtcctctttt
agagcagcaa gtatttccag ccacttagtc acctctgcag ccccttattt tcacagtctt
gagacaagaa tctcactctt tagcccatat tggcctggaa ctttaggcag tcctgccgga
gtttcagact gctgggatga caggcctgac ccattacgtc cactaaggat ggtttccttt
cctgtgagct agcagcatgt agactccaca aggctcctgg ggaagtgttg ttatagtatg
ttatagtata gttgcgaaag gaaggttttc agaagatatg ggtattacga agaaattcta
tgtaaagttt cttttggatt ctctgtttgt atAGATCCAG CATGTCTGGC TTGCACCTAG
TAAAACAAGG TCGAGACAGA AAGAAAATAG ACTCACAACG AGATTTCACT GTGGCTTCTC
CAGCAGAATT TGTTACTCGT TTTGGGGGAA ATAAAGTAAT TGAGAAGgta agttaaactt
actaaactat ttcgcttgaa gtatgtgaga tttcatgcct agatttgttg tttctgttca
aaaggatatt taggttttta gggactttgc ctttttatgc agggctatcc tttctgtctc
cctagcatgt tactaataca taatctcact gtgtacctgt gtttttacat
In "Step 2" I selected:
Mus musculus- Mouse- UCSC Dec. 2011 (GRCm38/mm10)...
In "Step 3" I selected:
TTTN-23bp - Cpf1 Acidaminococcus / Lachnospiracea
My questions are as follows:
- Should I do anything differently to use the CRISPOR tool for my goals?
- Are there other free webtools available for me to achieve the goals I outlined above?
UPDATE: I emailed the folks at CRISPOR, and they informed me the site had been down, but they have since restarted it. Hopefully, their restart is sufficient, but I will leave my question up with the hopes that others can point me to more webtools.