PrimeDesign software for rapid and simplified design of prime editing guide RNAs

Abstract

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.

Fig. 1. PrimeDesign web application.

a PrimeDesign takes a single sequence as input encoding both the original reference and desired edited sequences, b recommends a candidate pegRNA and ngRNA combination to install the edit of interest, c provides sequence visualizations of the edit of interest, selected pegRNA and ngRNA designs, and predicted pegRNA secondary structures, and d enables the interactive design of both pegRNAs and ngRNAs that can be downloaded as a summary table.

Fig. 2. PrimeDesign analysis of the ClinVar database.

a The distribution of the number of designed pegRNA spacers per ClinVar variant. Candidate pegRNAs were determined based on the requirement of RTT length <35 nt and the RT extension to have a minimum homology of 5 nt downstream of the edit. b The 63,710 (91.7%) targetable ClinVar variants classified by type. The inner ring (gold) represents the proportion of targetable variants by type where at least one pegRNA could be designed to disrupt the PAM sequence (dark gold). The outer ring (gray) represents the proportion of targetable variants by type where at least one ngRNA could be designed for the PE3b strategy where the mismatch lies in the seed sequence (PAM-proximal nucleotides 1–10) (dark gray). See Supplementary Data 1 for details.

Fig. 3. Installation of human pathogenic variants in HEK293T cells with PrimeDesign.

a Overview of prime editing efficiencies for the installation of 20 human pathogenic variants in HEK293T cells, using PrimeDesign recommendations. Desired edit refers to sequencing reads containing only the edit of interest, while byproduct refers to sequencing reads containing any mutation(s) outside of only the edit of interest (i.e. indels, desired edit and indels). b Comparison between PE3 and PE3b editing strategies. c Assessing the effects of PAM-disrupting silent mutations on prime editing efficiencies. Mean ± s.d. of n = 3 independent biological replicates. Some of the data shown in a are also represented in b and c. See Supplementary Data 2 for details. Source Data is available in the Source Data file.