ACEofBASEs (a careful evaluation of BaseEdits)

Help on


  • name: provide a descriptive name for your search. This name will be used for the output files to download so that you can keep track of different searches.
  • Select either single query or batch search: in the first case, the sequence can be up to 500 nucleotides long. Paste a plain sequence, not fasta format. Only characters representing valid nucleotides (A,a,C,c,G,g,T,t,N,n) will be considered and any other character will be discarded. In the second case you can provide a (multi-)fasta file with any number of sequences and a total size of up to 500KB.
  • Base editor:

Target selection
  • target site 5’ limitation: typical in vitro transcription promoters, like the T7 or the U6, require one or two leading ‘G’ respectively. However, we and others [Hwang et al., Ansai & Kinoshita] found that adding one or two Gs to the desired target sequence can also give valid sgRNAs that induce double strand breaks (DSBs). This release of restriction identifies many more target sites in the query sequence (hence ‘NN’ is the default value).
  • target site 3’ limitation: for C. elegans it has been shown that ‘GG’ at the 3’ site enhances the introduction of a DSB [Farboud & Meyer]. However, as with the previous field, you can specify any valid sequence of two bases to restrict the list of possible CRISPR/Cas9 target sites.
  • 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 repective primers.

Off-target prediction
  • max. total mismatches: can be set to 0-5. Note: more than four mismatches in total prevent DSB induction [Hsu et al., Cho et al.].
  • max. core length: Mismatches at a distance to the PAM, will still allow the introduction of a DSB, while mismatches close to the PAM will abolish the introduction of a DSB [Hsu et al., Cong et al., Stemberg et al.]. The core is a simplified parameter to account for these findings and is defined as the nucleotides adjacent to the PAM (12 by default, can be set to 2-20). The checkbox in front allows to enable/disable the core parameters. The prediction of off-target sites taking into account the core is only available when using the PAM motif from S. pyogenes. In the other cases there is not yet experimental evidence suggesting that the core can be benefitial.
  • max. core mismatches: can be set to 0-2. Note: in the core, more than 2 mismatches abolishes DSB introduction at the potential off-target position [Hsu et al., Cong et al.]

    Cas9-sgRNA complex
  • species: define in which genomic context off-targets should be predicted.

Output

TBC

References

  • Ansai, S. & Kinoshita, M. Targeted mutagenesis using CRISPR/Cas system in medaka. Biol. Open 3, 362–71 (2014).
  • Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–41 (2014).
  • Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science (2013). doi:10.1126/science.1231143
  • Farboud B. & Meyer B. J. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design. Genetics (2015).
  • Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–32 (2013).
  • Hwang, W. Y. et al. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8, e68708 (2013).

Access to the results

The results produced by ACEofBASEs will be available online for a period of time depending on the type of search after the seach job has finished, either successfully or not:

  • Single search: 48 hours
  • Batch search: 7 days
After this time any result will be deleted from our system. The users are advised to download their results as soon as possible. The aforementioned times can be reduced due to internal restrictions.

Citing this tool

If you use this tool for you scientific work, please cite it as:
Cornean A, Gierten J, Welz B, Mateo JL, Thumberger T, Wittbrodt J. Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction. Elife. 2022 Apr 4;11:e72124. doi: 10.7554/eLife.72124.

Recommendation:

This tool is aimed at helping researchers to use the CRISPR technology. However, there are many factors that can influence the final result of a CRISPR experiment. Therefore, we recommend check carefully the results of the experiments in a independent manner.

Feedback:

If you encounter any problem using this tool or you have any suggestion, please email us:
ed.grebledieh-inu.soc@rpsirc.