CCTop - CRISPR/Cas9 target online predictor
name:
single query batch mode
query sequence: (plain nucleotide sequence, max 500 bases)
(multi-)fasta file
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PAM type
Select the PAM motif corresponding to the CAS system of your choice.
:
Target selection Off-target prediction

     ┌┬5′ limitation     ┌─┬PAM
  5' NNNNNNNNNNNNNNNNNNNNNGG
          3′ limitation┴┘



target site length
Define the length of the target size, sequence upstream of the PAM.
:
target site 5' limitation
(In vitro) transcription depends on a leading 'G' (U6 promoter) or 'GG' (T7 promoter). However, it was shown that the leading guanin(s) can also be added or substituted at the 5' end of any sgRNA target site, hence 'NN' is set as default.

Hwang et al., PLoS One, (2013)
Ansai & Kinoshita, Biol. Open (2014)
:
target site 3' limitation
A recent paper in C. elegans demonstrated that a ‚GG’ motif adjacent to the PAM could be beneficial for DSB introduction.

Farboud & Meyer, Genetics (2015).
:

In vitro transcription
Select your in vitro transcription method. We will provide the forward and reverse oligos to clone each candidate sgRNA into the appropriate vector. If you select "Custom" you can specify your overhangs, which will be appended to the 5' of the sgRNA sequence.
T7 U6 Custom
fwd overhang:
rev overhang:

     ─────max 4 MM─────┌─┬PAM
  5' NNNNNNNNNNNNNNNNNNNNNGG
             ──────────
                    max 2 MM core


max. total mismatches
Maximum number of mismatches taken into account for prediction of potential sgRNA off-target sites. Note: more than four mismatches in total will most likely prevent DSB induction.

Hsu et al., Nat. Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:
adjust mismatch values!

core length
Check to enable the 'core parameter'.

More than two mismatches in the first 8-14 nucleotides adjacent to the PAM were demonstrated to abolish Cas9 mediated DSB introduction. The 'core' is a simplified parameter to account for these findings.

Cong et al., Science (2013)
Hsu et al., Nat. Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:

max. core mismatches
Maximum number of mismatches to be allowed in the core region of potential sgRNA off-target sites. Note: more than two mismatches in total will most likely prevent DSB induction.

Hsu et al., Nat. Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:
adjust mismatch values!

species
Choose a reference genome for sgRNA target site evaluation.
:




If you use this tool for your scientific work, please cite it as:

Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J. and Mateo, J.L. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLOS ONE (2015). doi: 10.1371/journal.pone.0124633