Abstract
The success of CRISPR-mediated gene perturbation studies is highly dependent on the quality of gRNAs, and several tools have been developed to enable optimal gRNA design. However, these tools are not all adaptable to the latest CRISPR modalities or nucleases, nor do they offer comprehensive annotation methods or scalability for advanced CRISPR applications. Here, we present a new ecosystem of R packages that enables efficient gRNA design and annotation for a multitude of CRISPR technologies, including CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi) and CRISPR base editing (CRISPRbe). The core package, crisprDesign, offers a comprehensive, user-friendly, and unified interface to add on- and off-target annotations via several alignment methods, rich gene and SNP annotations, and a dozen on- and off-target activity scores. These functionalities are enabled for any RNA- or DNA-targeting nucleases, including Cas9, Cas12, and Cas13. We illustrate the general applicability of our tools by designing optimal gRNAs for three case studies: tiling CRISPRbe library for BRCA1 using the base editor BE4max, tiling RNA-targeting libraries for CD46 and CD55 using CasRx, and activation of MMP7 using CRISPRa. Our suite of R packages is open-source and deployed through the Bioconductor project to facilitate their use by the CRISPR community.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Fixed typos
Abbreviations
- ABE
- adenine base editor
- BWA
- Burrows-Wheeler Aligner
- CAGE
- Cap Analysis of Gene Expression
- CBE
- cytosine base editor
- CCLE
- cancer cell line encyclopedia
- CDS
- coding DNA sequence
- CFD
- cutting frequency determination
- CRISPR
- clustered regularly interspaced short palindromic repeats
- CRISPRa
- CRISPR activation
- CRISPRbe
- CRISPR base editing
- CRISPRi
- CRISPR interference
- DHS
- DNAse I hypersensitive site
- DSB
- DNA double-strand break
- FACS
- fluorescence-activated cell sorting
- lncRNAs
- long non-coding RNAs
- MAF
- minor allele frequency
- OPS
- optical pooled screening
- PAM
- protospacer adjacent motif
- PE
- prime editing
- pegRNA
- prime editing guide RNA
- PFS
- protospacer flanking sequence
- RNAi
- RNA interference
- gRNA
- single-guide RNA
- shRNA
- short-hairpin RNA
- siRNA
- small-interfering RNA
- SNP
- single-nucleotide polymorphism
- TSS
- transcription starting site