PROpeg Comprehensive Tutorial

PROpeg is a state-of-the-art computational platform tailored for designing and analyzing prime editing guide RNAs (pegRNAs) in plant systems.

PROpeg Quick Start Video Tutorial

Step 1: Inputting Your Sequences

Begin by providing the DNA sequences in the Guide Design tab.

  • Wildtype/Reference Sequence: The original, unmodified DNA sequence.
  • Edited/Desired Sequence: The DNA sequence containing your intended mutation.
  • One sequence per run: PROpeg designs a single target at a time. Provide one wildtype and one edited sequence; uploaded files must contain a single sequence. Batch / multi-FASTA input is not supported.
  • Load Example 1: Auto-fills sequences where machine-learning efficiency prediction is OFF due to insufficient flanking context (less than 99 bp).
  • Load Example 2: Auto-fills sequences where prediction is ON because it satisfies the 99 bp upstream and downstream requirement.
Tip: For the predictions along with pegRNAs, ensure your target edit has at least 99 base pairs upstream and downstream of the edit.

Step 2: Configuring Parameters

Under the Parameters tab, you can fine-tune your prime editing system:

  • PAM Sequence: Choose from NGG, NG, or a Custom user-defined sequence.
  • Cut distance to PAM: Define the cleavage position offset (default -3).
  • Spacer length: (Range 1-40) Adjust the standard spacer length.
  • Spacer GC content (%): Constrain GC pairs in the spacer sequence (0-100%).
  • Prime editing window: Focus the edit inclusion bounds (1-15).
  • PBS length: Set exact lengths (7-16) for the PBS.
  • PBS GC content (%): Define GC boundaries (0-100%) for the PBS.
  • Recommended Tm of PBS sequence: Directly control the melting temperature (default 30°C).
  • Homologous RT template length: (Range 7-16) Adjust the RT template size.
  • Toggle Options: Enable optional design models:
    • Tm-directed PBS length model — size the PBS by melting temperature rather than fixed length.
    • Dual-pegRNA model — process a paired-pegRNA strategy where supported.
    • Exclude first C in RT template — avoid a 5' C at the start of the RT template.
    • PE3 / PE3b secondary nicking — add a nicking sgRNA on the non-edited strand within your chosen nick-to-nick distance range, preferring an edit-specific PE3b nick when available to boost efficiency.

Step 3: Primer Design

Under the Primer tab, configure the primers required for the assembly of your pegRNA expression vectors:

  • Pre-configured Primer Types: Instantly select standard plant editing system architectures like pOsU3, pTaU3, pTaU6, or pH-nCas9-PPE-V2 to auto-load the necessary primers.
  • Custom Primers: Alternatively, choose Custom to explicitly define the left and right parts of your Forward and Reverse primers manually.

Step 4: Interpreting Results & Structure Analysis

Depending on your application, click one of the four design buttons: Design pegRNA, Design g-pegRNA, Design epegRNA, or Design g-epegRNA. The algorithm then calculates permutations against thermodynamic boundaries and scoring models.

  • Program & Recommendation Rows: Results are systematically grouped into programs (highlighted in green). Within each program grouping, the most thermodynamically and functionally optimal PBS and RT template parameters for a design are designated as Recommended! (highlighted in red).
  • Column Features: Each row explicitly delineates the designed sequences for the Spacer-PAM, Linker, PBS, and RT Template, as well as indicating the target sequence Strand orientation (Sense/Antisense).
  • Efficiency Score: Predicted editing efficiency, utilizing a varaiant of deep-learning tool algorithm (PRIDICT2.0), adopting the baseline (HEK293T) score.
  • Secondary Structure Visualization: The results table will feature a specific visualization button matching your chosen design (pegRNA, g-pegRNA, epegRNA, or g-epegRNA).
    • pegRNA: Visualizes the standard folded RNA string, showcasing spacer, scaffold, RT, and PBS sections correctly aligned.
    • g-pegRNA: Highlights the specific modification of the last three nucleotides of the 86-nucleotide scaffold sequence, demonstrating exact dot-bracket base pairing.
    • epegRNA: Engineered pegRNA — attaches a 3' protective structured motif (tevopreQ1 sequence) to the RT template through an optimal linker sequence, on the standard (unmodified) scaffold, to resist exonuclease degradation.
    • g-epegRNA: Combines the g-pegRNA scaffold modification with the epegRNA 3' motif — an optimal linker sequence attaches the tevopreQ1 motif on top of the modified scaffold, enhancing both editing purity and transcript stability in vivo.

Frequently Asked Questions