This code designs every possible guide in the CDS region, extending 30 nucleotides into intronic and UTR regions for user-defined transcripts. It then annotates the possible edits for each guide. In its original implementation, amino acid change predictions were generated for single base edits; however, for certain codons (e.g., CCC encoding Proline), the tool does not explicitly account for cases where multiple adjacent cytosines may be edited simultaneously. To address this, a custom function was added to predict these additional mutation outcomes. Separate files annotating ClinVra SNPs are also generated.
Mudra Hegde, Ruth Hanna
Email: mhegde@broadinstitute.org, rhanna@broadinstitute.org
Version: 2.0 (debugged and updated)
Contributors: Murugesh Narayanappa (mnarayan@stanford.edu) (debugging and bug fixes)
Input File:
.txt file with list of Ensembl transcript IDs in the first column and gene symbols in the second column OR FASTA file with nucleotide sequence
Variant File:
File with ClinVar SNPs (variant_summary.txt). This file can be downloaded from:
ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/tab_delimited/variant_summary.txt.gz
Input type:
Indicate whether the file contains a list of transcripts or a nucleotide sequence
Base editor type:
Indicate the type of base editor (Rees et al., 2018) for which designs are required. This choice dictates the choice of PAM, sgRNA length, editing window, and type of edit
PAM:
PAM preference if BE type has not been selected; Default: NGG
Edit window:
Editing window relative to nucleotide position in sgRNA, if BE type has not been selected; Default: 4–8
sgRNA length:
Length of sgRNA excluding PAM sequence, if BE type has not been selected; Default: 20
Edit:
Type of edit made by base editor, if BE type has not been selected; Default: all (annotates both C→T and A→G edits)
Intron buffer:
Number of bp into the intron to consider for guide design
Filter GC:
Whether to filter out edits in a GC motif
Output name:
Name for output folder
A custom function has been added to account for previously missed mutations. Please refer to the .Rmd file, which includes both the code and an example.
Purpose: Identify and append these missed dual-edit mutations and their categories to the output dataframe. Background: The tool predicts amino acid changes by evaluating one nucleotide at a time.However, when a codon contains “AA” or “CC” in the first two positions (before the wobble base), in either strand orientation (5’→3’ or 3’→5’) the base editor is likely to edit both nucleotides simultaneously: Adenine Base Editor (ABE): AA → GG Cytosine Base Editor (CBE): CC → TT Since the tool only evaluates single-nucleotide edits, it does not predict the amino acid outcomes resulting from these probable dual-edit events by the base editor.