pegFinder: A pegRNA designer for CRISPR prime editing

Abstract

To the EditorCRISPR technologies have been widely adopted as powerful tools for targeted genomic manipulation 1. Recently, a new CRISPR-based strategy for precision genome editing was developed that enables diverse genomic alterations to be directly written into target sites without requiring double-strand breaks (DSBs) or donor templates 2. Termed prime editing, this approach involves two key components: 1) a catalytically impaired Cas9 nickase fused to a reverse transcriptase (PE2), and 2) a multifunctional prime editing guide RNA (pegRNA) that specifies the target site and further acts as a template for reverse transcription (RT). pegRNAs are similar to standard single-guide RNAs (sgRNAs), but additionally have a customizable extension on the 3 end. The 3 extension is composed of a RT template that encodes the desired edit and a primer binding site (PBS) that anneals to the target genomic site to prime the RT reaction 2. These additional components considerably increase the complexity of pegRNA design compared to standard sgRNAs. While many tools have been developed for identifying candidate sgRNAs in a target DNA sequence 3-8, no user-friendly web application currently exists for designing pegRNAs. We therefore developed pegFinder, a streamlined web tool that rapidly designs candidate pegRNAs (Figure 1). The pegFinder web portal is freely available at http://pegfinder.sidichenlab.org/ (Supplementary Figure 1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/081612v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1494188org.highwire.dtl.DTLVardef@6d80ccorg.highwire.dtl.DTLVardef@122eaf1org.highwire.dtl.DTLVardef@16e8e71_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO pegFinder: A pegRNA designer for CRISPR prime editing Schematic of the pegFinder workflow for designing CRISPR prime editing pegRNAs. The user provides the wildtype DNA sequence, and the edited DNA sequence. Optionally, the user can include the results from sgRNA designer tools to pegFinder, or specify a preselected sgRNA spacer to be used for pegRNA design. pegFinder then generates a pegRNA that can be used to engineer the desired alterations. pegFinder also reports alternative RT templates and PBS sequences of varying lengths that can be swapped into the pegRNA for downstream experimental optimization. pegFinder further reports secondary nicking sgRNAs that can increase prime editing efficiency (PE3 method). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/081612v1_figS1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@149ba2corg.highwire.dtl.DTLVardef@15da377org.highwire.dtl.DTLVardef@96fe3borg.highwire.dtl.DTLVardef@10cfee0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSupplementary Figure 1:C_FLOATNO pegFinder web interface Screenshot of the pegFinder web interface, available at http://pegfinder.sidichenlab.org. C_FIG