My goals are to use a free webtool to:

  1. 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.
  2. 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  
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:

  1. Should I do anything differently to use the CRISPOR tool for my goals?
  2. 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.

  • $\begingroup$ Crispr tech. is very cool. Was wondering if you wouldn't mind explaining a little bit a bout the technology and your process described above? $\endgroup$ – user7875084 May 7 '17 at 12:12

Most online tools will only help you to design gRNAs for Cas9 because it is the one that is most commonly used. I found an online suite called RGEN tools that also has an option for AsCpf1.

However, if you don't find it satisfactory then my suggestion is that you either design your gRNA manually or write your own script (which won't be that complex).


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