GeneTargeter

Creates custom gene-editing constructs designed to deliver DNA payload to specific genes in malaria parasites, including for knock-out or conditional knock-down in Plasmodium falciparum.

Preprint available on ResearchSquare. Command line interface and code available through GitHub. Developed by the Niles Lab at MIT.


Choose a pre-loaded targeting system:  

or


(Download a custom base plasmid example here)

Target location within gene:  


Guide RNAs will be selected automatically based on Doench et al. (2016) on-target score and either Doench et al. (2016) or Hsu et al. (2013) off-target scores. Off-target scores are evaluated based on off-target gRNAs in the P. falciparum 3D7 genome.

Alternatively, you may manually annotate all CRISPR guide RNAs to be included in targeting. Guide RNA annotations should start with the word "gRNA" in the label, followed by a number indicating their order of preference. You may also annotate hand-picked left and right homologous regions on your file (annotated with the words "LHR" and "RHR" in the label, respectively), if you do not want GeneTargeter to choose them automatically for you.


Manual HR/sgRNAs

Use user-annotated homology regions and/or guide RNAs (if any):

Bulk processing options:

Consolidate files by type:   Project prefix:   Start numbering on:

Treat genes as coding/noncoding:  


Right chromosomal integration homologous region

Size:
   Minimum:    bp
   Preferred: bp
   Maximum:    bp

Maximum distance from gene: bp

Search for best start/end point bp upstream and bp downstream of first valid start/end point

Minimum melting temperature of the first and last bp of the right homologous region: ºC

Left chromosomal integration homologous region

Size:
   Minimum:    bp
   Preferred: bp
   Maximum:    bp

Maximum distance from gRNA: bp

Search for best start/end point bp upstream and bp downstream of first valid start/end point

Minimum melting temperature of the first and last bp of the left homologous region: ºC

Gibson Assembly

Annealing region size:    Minimum:    bp
   Preferred: bp
   Maximum:    bp

*Gibson homology region is taken as above preferred size.

Minimum primer melting temperature: ºC

Maximum melting temperature difference between primers: ºC

Guide RNA (gRNA) Selection

Minimum GC content:   %
CRISPR cutting enzyme:
PAM Sequence:  
On-target scoring method:
Minimum on-target score:   %
Off-target scoring method for single hits:

Minimum off-target total score:   %
Maximum single hit off-target score:  %



Recoded Region

Codon optimize to using

Minimum gBlock size: bp

Maximum gBlock size: bp

Extend gBlock if under minimum size (instead of using oligos):

Use HA tags (5' designs):





Provide a list of 3D7 gene IDs or upload a GenBank file annotated with the gene's sequence in 5'->3' sense. If you load your own files, make sure the target gene name in each text box matches the gene's annotation on the plasmid.

or

GeneTargeter outputs seven kinds of files:

On-target Cas9 gRNA scoring code and CFD scoring matrix obtained December 27, 2016 from Doench et al. (2016) supplementary material.

On-target Cas12 gRNA scoring coefficients obtained from Kim et al. (2017) supplementary material, equivalent to the CINDEL online tool. Exact on-target scores may vary between GeneTargeter and CINDEL due to minor differenes in gRNA free energy calculations.

Self-folding Gibbs free energy of gRNAs (used to calculate Kim et al. (2017) CINDEL scores) calculated using RNAfold obtained from ViennaRNA v2.3.3 (Lorenz et al., 2011).

Off-target Zhang Lab scoring matrix obtained December 27, 2016 from the online MIT CRISPR tool (Hsu et al., 2016).

Off-target gRNA database built from P. falciparum genome first published by Gardner et al. (2002) and subsequently edited by the PlasmoDB community.

Codon frequency tables obtained from the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUT) database (Athey et al., 2017).

Niles lab logo design by Gaël Chambonnier.

Background Image: Plasmodium falciparum in a patient presenting with pyrexia and headache. O. Erhabor (2013)
CC BY-SA 3.0



All other code: MIT License, appropriately.
GitHub src here.
Developed by Pablo Cárdenas Ramírez
pablocarderam@gmail.com