protoSpaceJAM

protoSpaceJAM automates and optimizes gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments across multiple Cas enzymes.

Key Features:

• gRNA Ranking: Utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions.
• Recoding Mutations: Introduces silent mutations and mutations in non-coding sequences in HDR donors to prevent re-cutting and increase knock-in efficiency.
• Donor Customization: Supports design of double-stranded or single-stranded donors with user-customizable parameters.
• Genotyping Primer Design: Includes an additional module for designing genotyping primers for deep sequencing of edited alleles.
• Cas Enzyme Support: Currently supports SpCas9, SpCas9-VQR, and enAsCas12a.

Scientific Applications:

• Precise Genome Engineering: Enables CRISPR/Cas-mediated knock-in of DNA sequences for research and therapeutic applications.
• Knock-in Efficiency Optimization: Increases knock-in efficiency through recoding mutations and optimal strand selection for single-stranded donors.
• Edited Allele Validation: Facilitates deep sequencing of edited alleles through integrated genotyping primer design.

Methodology:

protoSpaceJAM applies biological rules to rank gRNAs and incorporates recoding mutations in HDR donors; validation demonstrated increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors.