PrimeDesign

Reference

• paper : PrimeDesign software for rapid and simplified design of prime editing guide RNAs(Nat Commun . 2021 Feb 15;12(1):1034. doi: 10.1038/s41467-021-21337-7.)
• github : https://github.com/pinellolab/PrimeDesign
• web tools :https://drugthatgene.pinellolab.partners.org/

web application

input 2nd structure
output

Input

• Substitution: (/)
• Insertion: (+)

• Deletion: (-)

  
  image.png
  input

Parameters

• PBS length
• RTT length
• Nicking distance
• Remove extension with C start
• homopolymer T stretch
• Disrupt PAM with silent PAM mutation
• Calculate CFD score

Output

---

Pooled design

(https://drugthatgene.pinellolab.partners.org/pooled)

pooled

Input

Genome-wide

input_example_pooled

Saturation mutagenesis

Saturation mutagenesis

• Saturation mutagenesis type
  • Base substitutions
  • Amino acid substitutions

Output

output

---

Command line tool

Installation

docker pull pinellolab/primedesign

Usage

docker run -v ${PWD}/:/DATA -w /DATA pinellolab/primedesign primedesign_cli [options]

-f \
-pbs \
-rtt \
-nick_dist_min -nick_dist_max\
-filter_c1 \
-sat_mut \
-n_pegrnas \
-nick_dist_pooled \
-pbs_pooled \
-rtt_pooled \
-out 

Users can specify the following options:

-f, --file
      Input file (.txt or .csv) with sequences for PrimeDesign. Format: target_name,target_sequence (column names required)

-pbs, --pbs_length_list
      List of primer binding site (PBS) lengths for the pegRNA extension (Default: 10 to 15 nt). 
        Example: 12 13 14 15
-rtt, --rtt_length_list
      List of reverse transcription (RT) template lengths for the pegRNA extension (Default: 10 to 30 nt). 
        Example: 10 15 20
-nick_dist_min, --nicking_distance_minimum
      Minimum nicking distance for designing ngRNAs upstream and downstream of a pegRNA 
        (Default: 0 bp).
-nick_dist_max, --nicking_distance_maximum
      Maximum nicking distance for designing ngRNAs upstream and downstream of a pegRNA 
        (Default: 100 bp).
-filter_c1, --filter_c1_extension
      Option to filter against pegRNA extensions that start with a C base.
-silent_mut, --silent_mutation
      Introduce silent mutation into PAM assuming sequence is in-frame. Currently only available with SpCas9.
-genome_wide, --genome_wide_design
      Whether or not this is a genome-wide pooled design. This option designs a set of pegRNAs per input without ranging PBS and RTT parameters.
-sat_mut, --saturation_mutagenesis
      Saturation mutagenesis design with prime editing. The 'aa' option makes amino acid changes and the 'base' option makes DNA base changes. (Options: aa, base)
-n_pegrnas, --number_of_pegrnas
      The maximum number of pegRNAs to design for each input sequence. The pegRNAs are ranked by 1) PAM disrupted > PAM intact then 2) distance to edit. 
        (Default: 3)
n_ngrnas, --number_of_ngrnas
      The maximum number of ngRNAs to design for each input sequence. The ngRNAs are ranked by 1) PE3b-seed > PE3b-nonseed > PE3 then 2) deviation from nicking_distance_pooled. 
        (Default: 3)
-nick_dist_pooled, --nicking_distance_pooled
      The nicking distance between pegRNAs and ngRNAs for pooled designs. PE3b annotation is priority (PE3b seed -> PE3b non-seed), followed by nicking distance closest to this parameter. 
        (Default: 75 bp)
-homology_downstream, --homology_downstream
      For pooled designs (genome_wide or saturation_mutagenesis needs to be indicated), this parameter determines the RT extension length downstream of an edit for pegRNA designs. 
        (Default: 10)
-pbs_pooled, --pbs_length_pooled
      The PBS length to design pegRNAs for pooled design applications. 
        (Default: 14 nt)
-rtt_pooled, --rtt_max_length_pooled
      The maximum RTT length to design pegRNAs for pooled design applications. 
        (Default: 50 nt)
-out, --out_dir
      Name of output directory. (Default: ./DATETIMESTAMP_PrimeDesign)