| name | bioconductor-crisprdesign |
| description | Provides a comprehensive suite of functions to design and annotate CRISPR guide RNA (gRNAs) sequences. This includes on- and off-target search, on-target efficiency scoring, off-target scoring, full gene and TSS contextual annotations, and SNP annotation (human only). It currently support five types of CRISPR modalities (modes of perturbations): CRISPR knockout, CRISPR activation, CRISPR inhibition, CRISPR base editing, and CRISPR knockdown. All types of CRISPR nucleases are supported, including |
crisprDesign
Workflows
Standard Workflow
library(crisprDesign)
library(BSgenome.Hsapiens.UCSC.hg38)
data(grListExample, package="crisprDesign")
data(BE4max, package="crisprBase")
bsgenome <- BSgenome.Hsapiens.UCSC.hg38
gr <- queryTxObject(txObject=grListExample, featureType="cds", queryColumn="gene_symbol", queryValue="IQSEC3")
gr <- gr[1]
guideSet <- findSpacers(gr, bsgenome=bsgenome, crisprNuclease=BE4max, strict_overlap=FALSE)
Inputs: GRangesList gene coordinates (grListExample), BSgenome object (bsgenome), and a BaseEditor/CrisprNuclease object (BE4max). Output: A GuideSet object containing designed spacer sequences.
When to Use
- CRISPR Base Editing Design: Designing and characterizing gRNAs for base editors (e.g., cytidine base editor BE4max) using
findSpacers and predicting edited alleles with addEditedAlleles.
- Gene Feature Querying: Querying transcript objects for specific features (e.g., coding sequences) using
queryTxObject.
- Transcript Annotation Retrieval: Obtaining transcript-specific annotations for predicted allele analysis using
getTxInfoDataFrame.
When NOT to Use
- Unsupported R/Bioconductor Versions: Do not use if R version is less than 4.2.0 or Bioconductor version is less than 3.16.
- General Sequence Alignment: For general sequence alignment tasks where specialized aligners (like Bowtie/BWA directly) are more appropriate.
Data Requirements
- Gene Coordinates: A
GRangesList object containing gene coordinates (e.g., grListExample).
- Reference Genome: A
BSgenome object containing reference genome sequences (e.g., BSgenome.Hsapiens.UCSC.hg38).
- Nuclease/Base Editor Specification: A
BaseEditor or CrisprNuclease object (e.g., BE4max or SpCas9).
Key Parameters
- txObject (no default): A
GRangesList object containing gene model coordinates used in queryTxObject and getTxInfoDataFrame.
- featureType (no default): Character string specifying the gene feature to query (e.g.,
"cds").
- crisprNuclease (no default): A
CrisprNuclease or BaseEditor object specifying the CRISPR enzyme.
- strict_overlap (
TRUE): Logical indicating whether spacer sequences must strictly overlap the target region.
- editingWindow (NULL): Numeric vector of length 2 specifying the window of editing relative to the PAM site.
- minEditingWeight (
0): Minimum editing weight required for an allele to be predicted.
- minMutationScore (
0.3): Minimum predicted probability for labeling an allele with a predicted variant.
Best Practices
- Feature Extraction: Use
queryTxObject to extract specific genomic features (like "cds") before running spacer design.
- Flexible Overlaps: Set
strict_overlap = FALSE in findSpacers for base editing design to allow the editing window to extend beyond the protospacer sequence region.
- Window Limitation: Limit the
editingWindow size in addEditedAlleles to avoid exponential increases in computing time.
Common Pitfalls
- Large Editing Windows: Providing a very large
editingWindow to addEditedAlleles exponentially increases computing time. Fix: Use a narrower window like c(-20, -8).
- Strict Overlaps in Base Editing: Using
strict_overlap = TRUE for base editing might miss spacers whose editing window extends outside the target region. Fix: Set strict_overlap = FALSE.
Alternatives
- crisprBase: For core CRISPR functions and S4 objects.
- crisprBowtie: For aligning gRNA spacers to genomes using bowtie.
- crisprBwa: For aligning gRNA spacers to genomes using BWA.
- crisprScore: For calculating on- and off-target scores.
- crisprViz: For visualizing gRNAs using genomic tracks.
Citations
- Koblan, Luke W, et al. 2018. "Improving Cytidine and Adenine Base Editors by Expression Optimization and Ancestral Reconstruction." Nature Biotechnology 36 (9): 843–46.
References
- Homepage: bioconductor.org/packages/crisprDesign
- Vignette: bioconductor.org/packages/release/bioc/vignettes/crisprDesign/inst/doc/base_editing.html
