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, 283 (17), 3232-8

CRISPR Guide RNA Design for Research Applications


CRISPR Guide RNA Design for Research Applications

Stephanie E Mohr et al. FEBS J.


The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state of the art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation, and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics.

Keywords: CRISPR-Cas9; CRISPRa; CRISPRi; bioinformatics; gene editing; genome annotation; genome engineering.


Figure 1
Figure 1
Workflow for batch‐mode design of gRNAs for CRISPR knockout. Starting with a set of all possible gRNA designs based on an appropriate adjacent PAM sequence, gRNAs are first ranked based on various criteria, then filtered to enrich for or avoid specific features. When a gene is not covered with the most stringent set of criteria, it re‐enters the pipeline for design with relaxed criteria applied. Notably, if gene annotations are updated between the time of library design and experimental data analysis, interpretation of off targets, isoform coverage, etc. should be updated based on the current annotation in order to facilitate the most up‐to‐date interpretation of data.

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