Crisprdesign

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