How to use BEscreen

We're happy you found your way to BEscreen. BEscreen lets you easily design base editing libraries for screens of pre-specified variants. It can also generate libraries to introduce any possible edit in a given gene, transcript or genomic region (saturation screens). With BEscreen, you can also design guides that introduce an edit with a given consequence, such as guides that lead to missense, non-sense, splice-site edits etc. For example, this way, you can use BEscreen to design negative controls (non-editing or synonymously editing guides) for new and already existing libraries.

Of course you can also design individual guides for single edits. BEscreen accepts several input formats: if you want to screen for given variants ("variant mode"), you can input genomic variants, protein amino acid changes, and rsIDs from dbSNP. If you want to generate guides for a given genomic region (e.g., all coding sequences of a given gene; "gene/region mode"), you input gene names, transcript names or genomic regions.

Finally, BEscreen allows for comprehensive customization of your editing tools - select one of the pre-specified presets to set base editor options or input the characteristics of your base editor of choice (including customizing the editing window, fully customizable PAM site etc.).

To get started with BEscreen, follow the flow diagram to find out which mode you need to use and start designing your base editing library!

Decision flow chart BEscreen

Entering your data and options

This is how it looks:

The sidebar of BEscreen

Input selection

Variant(s)

If you want to perform a screen for a list of specific variants, select Variant(s): Given SNVs, AA changes, rsIDs as input. As the option indicates, BEscreen allows you to enter your variants as genomic variants (e.g., 12_6537866_C_T), as protein amino acid changes using gene or transcript names (e.g., GAPDH-L270F or GAPDH-201-L270F), or as an rsID (e.g., rs1062436) as outlined in more detail below. You can mix different input formats.

You can choose whether you want to directly enter the given variants in the corresponding field like this:

The variant input of BEscreen

or to upload a CSV file containg your variants of interest:

The variant input of BEscreen from CSV file

Direct variant inputs need to be provided either as:

genomic positions:
[chromosome]_[genomic_position]_[alternative_base] or [chromosome]_[genomic_position]_[reference_base]_[alternative_base]
e.g.: 12_6537866_C_T or 12_6537866_T
rsID:
e.g.: rs1062436
protein amino acid changes
[gene_symbol]-[transcript_number]-[WTAApositionMUTAA] or [gene_symbol]-[transcript_number]-[WTAApositionMUTAA]
e.g.: GAPDH-L270F or GAPDH-201-L270F
If you don't provide a transcript number the MANE select transcript will be used.

e.g. like this:

A variants input example of BEscreen

If you choose to input from file, you can use the columns variant or chr, pos, ref and alt. BEscreen will detect your columns automatically if you only provide one option. If your file contains all five columns, you need to specify which columns to use in the Input format field. The column variant can be populated by genomic positions (joined by underscores, rsIDs or protein amino acid changes). The columns chr, pos, ref and alt can only be populated by individual genomic positions.

Thus, CSV files containing variants need to be formatted as either of the following examples:

columns variant

variant
12_6537866_C_T
12_6537866_T
rs1062436
GAPDH-L270F
GAPDH-201-L270F

columns chr, pos, ref and alt:
```
chr,pos,ref,alt
12,6537866,C,T
```

The reference base can be omitted in any case (in the file you can leave the column out completely), but then BEscreen will not check if your reference base is the one it also finds. If these differ this indicates the usage of different reference genomes.

BEscreen will annotate the guides with several information. If you choose Use MANE select transcripts only, BEscreen will only use the MANE Select transcript for these annotations.

Gene(s)

If you don't want to start with pre-specified variants but rather want to design guides for a given gene or transcript, select Gene(s): Saturate whole CDS of genes. Next, select whether you want to input directly:

The genes input of BEscreen

or from a CSV file:

The genes input of BEscreen

Direct variant inputs need to be provided either as HGNC gene symbol (e.g., GAPDH) or transcript name (gene name followed by a hyphen and the transcript number; e.g., GAPDH-201) e.g. like this:

A genes input example of BEscreen

If you choose to input from file you can use the column symbol as shown in the following example:

columns symbol:
```
symbol
GAPDH
GAPDH-201
```

Take note that the input is case sensitive, so enter "GAPDH" for the human gene, but "Gapdh" for the mouse gene.

If you use a gene name as input, BEscreen will use all transcripts for this gene to search and annotate the guides. If you choose Use MANE select transcripts only, BEscreen will only use the MANE Select transcript to search and annotate the guides. This results in guides filtered for the MANE transcript.

Negative and positive controls for your assay

The Gene(s) mode has the important functionality to design proper positive and negative controls for base editing experiments.

Positive controls

Base editors can introduce edits that are equivalent (or mostly equivalent) to a gene knockout - stopgain and splice-site edits. The earlier one of those edits is introduced in your CDS, the more likely this will have the same consequence as a knockout of the gene. You can filter for those guides using BEscreen's Filter options.

Negative controls

Even more important than positive controls are negative controls. BEscreen allows for two types of negative controls that are well-suited for base editing screens: non-editing guides (i.e., guides that bind but do not confer any edits) and guides that only introduce a synonymous mutation (i.e., a base edit that leads to a synonymous codon).

Region(s)

If you neither want to start with pre-specified variants nor given genes, you can use a complete genomic region. To this end, select Region(s): Set a genomic region to scan for editable bases. Next, select whether you want to input directly:

The regions input of BEscreen

or from a CSV file:

The regions input of BEscreen

If you want to input your region(s) directly, you need to use the form [CHROM]:[START]-[END] (e.g. 12:6536490-6537490 like this):

A regions input example of BEscreen

If you choose to input from file you can use the column region as shown in the following example:

columns region:
```
region
12:6536490-6537490
```

Species

Next continue to the Species section and set your species by choosing a reference.

The species selection of BEscreen

Base editor options

Presets

Select a Preset from a list of known base editors. This will set your PAM and guide options. You can alter any setting made be the presets to adjust BEscreen to your needs, if you experienced different behavior in your system. If you do so, the preset field will show (changed) to show you that you are not using the original preset settings anymore.

PAM options

Either use presets from different Cas protein presets to set your PAM site sequence and PAM location or define a custom PAM sequence using IUPAC code and choose, if it is located at the 5' or 3' end of the guide. If you do the latter, the preset field will show (changed) to show you that you are not using any Cas protein preset settings anymore.

Guide options

You can adjust the Base change, Guide length and the Start and End of editing window in Guide options. Be aware that the editing window is well characterized for some base editors, but not so for others. In general editing windows in general are not to be considered absolute - edits outside of the editing window might always happen. To account for this you could either increase the editing window or use our safety region option to analyze the bases adjacent to the editing window.

The guide options of BEscreen

Filter options

If you are only interested in edits with specific consequences, you can set filters for synonymous_specific, splice-site, specific, missense, nonsense (stop gain), stop lost or start lost guides in the Filter options.

The filter options of BEscreen

Additional annotations

BEscreen provides feature-rich output, but if you need more, you can add the annotation of Ensembl's Variant Effect Predictor and genome wide hits using NCBI's BLAST.
For VEP, you can select which consequences to show (BEscreen defaults to pick the top consequence according to VEP's order for the option --pick) and you can set the shown fields as known from the command line version of this tool. Experimentally, you can also forward other command lines flags to VEP, but be warned again: This is an experimental feature that in some cases might lead to unexpected results.
For BLAST you can choose only to BLAST the main chromosomes without contigs like e.g. GL000218.1 and KI270728.1.

BEscreen offers additional annotations

Advanced options

Some more Advanced options are available. These differ for Variant(s), Gene(s) and Region(s). For all three you can set a safety region, if you know your base editor tends to edit outside its main editing window. BEscreen will not actively look for guides that edit in that region, but it will tell you, if the guides it found have an editable base in that area.

BEscreen's advanced options for all modes

For Variant(s) and Gene(s) there are additional options: