PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing

Abstract

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.

Graphical Abstract

Overviews for input and output of PE-Designer and PE-Analyzer.

Figure 1.

Schematic of a PE and the PE-Designer results panel. (A) Schematic of a PE, highlighting the pegRNA structure. (B) Interactive summary figure on the PE-Designer results page. The possible pegRNA target sequences are colored red and additional ngRNA sequences are colored blue. Each bar can be clicked to select that target sequence. When the amino acid sequence is changed, the altered residue is colored red. (C) Table of possible target sequences provided by PE-Designer. Checking the select button next to a target sequence displays a list of pegRNA extension sequences and additional ngRNA sequences. A red background means that the target is not recommended because the indicated parameter is unfavorable for prime editing.

Figure 2.

PE-Analyzer results panel. (A) Summary table indicating the number of sequence reads for each type of mutation. ‘Prime editing’ indicates the count of the desired sequence. (B) Bar graphs showing the distribution of insertions, deletions, and substitutions. The graphs can be changed by clicking the buttons above the graphs to switch the alignment of query sequences against either reference or expected sequences. (C) Table of alignment results. The results can be changed by clicking the buttons to switch the alignment of query sequences against either reference or expected sequences, or to filter the results by the mutation type; specific sequences can be found by entering that information.