HLAguide
This program is designed to generated all possible base-editor (ABE or CBE) guides against HLA alleles. Additional functionality has been added for Cas12-Detect design. This assumes a TTTV PAM, 20 nucleotide protospacer, and adds a single mutations in seed (1-5 proximal to PAM) to enhance specificity. For Cas12a, off-targets with >2 seed mutations and 1 seed & > 3 total mismatches are filtered out. For transparency all alleles are shown in “all alleles targeted by selected guide” tab with no filtering.
Please send any errors/questions to yuriy.baglaenko@cchmc.org
It can be run in four ways:
- Find all sgRNA that ablate an HLA allele - example - remove HLA-B27:05 while minimizing off-targets.
- Find all sgRNA that preserve an HLA alelle - example - remove everything
except HLA-B27:05 - Find all sgRNA that ablate an HLA allele and preserve an alternative allele - example - remove HLA-B27:05 but save HLA-B07:02.
- Cas12a Design for diagnostic purposes.
HLAguide, as part of a larger manuscript, will hopefully be submitted soon. bioarxiv link and publication link will be updated.
bioarxiv:
Step by step running instructions
- Select whether you want to ablate or preserve the chosen allele. Cas12 design is very separate and can be chosen here.
- Select a family (A / B / C etc )
- Input a 4 digit allele. Example: 07:02
- Input PAM and select a window of editing
- Optional: Alternative allele and population of interest are not required. Defaults to empty and all populations for downstream analysis and filtering.
- Click Run. This will autonavigate to the Predicted Guides tab.
- Calculations are completely in < a few minutes but please be patient. We’ve added a progress bar to provide clarity on steps.
- Click on a guide in any table and navigate to Plot or Primer tab to generate plots and primers.
- Feel free to navigate between tabs but remember, if you click on a new guide - the primers and plots will change.
A note on privacy
We do not track any inputs or outputs. All data are temporary and immediately deleted when the session is closed. We do, however, track anonymous time spent on the webpage. Each session is assigned a randomized ID with no links to IP or inputs.
Location of selected guide
Off target summary
Comparison of selected guide to alternative allele
Population frequency and cell line selector plots
Why are the off target and guide tables of different length?
Guides are repeated, ie ABE and CBE enzymes used to target donors and acceptors, and effeciencies are calcualted by enzyme. On the other hand, off-target analysis only uses the sequence as an input to identify other targeted alleles.
Why can’t I find sgRNA to my chosen target?
You’ve likely selected a 4 digit allele whose sequence cannot be pulled from IMGT. The software at the moment relies on genomic sequencing data deposited to IMGT. Future iterations may include the ability to upload your CDS sequence of choice to design only early stop codons.
My cell line plot is empty?
Sorry, no cell lines with the chosen HLA allele have been reliably identified in the CCLE/TCLP merged database.
Do you perform off-target predictions to the entire genome
No, we are focused on guides in the HLA. Off target prediction is only performed in the HLA region from annotated alleles deposited to IGMT.
What are the colors / annotations on the plots
A: Location of selected guide: color indicates orientation of guide. B: Comparions of selected guide to alternative allele: Red bar is the protospacer and PAM sequence. Protospacer is highlighted in green and PAM in red. Mismatched nucleotides are shaded orange. C: Cell line plots - the colors are different lineages. The x-axis is the other allele present in the cell (besides the one that was selected)
Are you going to release an accompanying R package.
I am working on it but the functionality will be minimal with no effeciency predictions or off target calculation. That will require integration with some of the software below. A github repo is in the works.
Citations
The work generates guides, ranks effeciencies, and calculates off-targets to HLA alleles. It is inspired by / adapts / and utilizes the works below. Please cite accordingly.
- IMGT/HLA : Barker, D. J. et al. The IPD-IMGT/HLA Database. Nucleic Acids Res 51, D1053-D1060 (2023).
- SpliceR : Kluesner, M.G., Lahr, W.S., Lonetree, Cl. et al. CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells. Nat Commun 12, 2437 (2021).
- iSTOP : Billon, P. et al. CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. Mol Cell 67, 1068-1079 e1064 (2017).
- FORECAST-BE : Pallaseni, A. et al. Predicting base editing outcomes using position-specific sequence determinants. Nucleic Acids Res 50, 3551-3564 (2022).
- BEdeep : Zhang, C. et al. Prediction of base editor off-targets by deep learning. Nat Commun 14, 5358 (2023).
- Cas-OFFinder : Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014).
- TCLP : Scholtalbers, J. et al. TCLP: an online cancer cell line catalogue integrating HLA type, predicted neo-epitopes, virus and gene expression. Genome Med 7, 118 (2015).
- CCLE : Ghandi, M. et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 569, 503-508 (2019). https://doi.org/10.1038/s41586-019-1186-3
- ARTEMIS : Kohabir, KAV. et al. Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS. Cell Rep Methods 16;4(12):100912 (2024).