What is LeishGEdit?

LeishGEdit (Leishmania Genome Editing) is an online resource for CRISPR Cas9 genome editing in kinetoplastids. On this site you can find plasmids and retrieve primer sequences to target your gene of interest for tagging and/or knockout.

imgWhat is new on LeishGEdit?

Contact

05 Aug 2020: Our paper "Bar-seq strategies for the LeishGEdit toolbox", previously published as a pre-print on BioRxiv, is now published in Molecular and Biochemical Parasitology.

19 Mar 2020: We have just added a new feature on LeishGEdit, allowing barcoded mutant screens as described in our previous study (Beneke et al., PLoS Pathogens 2019). We describe details for barcoding LeishGEdit primer in our manuscript on BioRxiv (Beneke and Gluenz, 2020).

23 Jan 2020: Check out our latest paper in the Journal of Cell Sciences on a new flagellar protein, which we termed LAX28.

26 Jun 2019: Our manuscript "Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections" is now available on PLoS Pathogens.

23 Nov 2018: Our latest manuscript "Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections" is now available for download. We use the power of our CRISPR-Cas9 toolbox to generate a knockout library of 100 mutants. We screen mutants for flagellar defects and use a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. Get it under: bioRxiv!

05 April 2018: We are running a CRISPR workshop for students from neglected disease endemic countries and the UK. Apply by 30th April. Course details can be found here: 2nd Advanced School in Genetic Manipulation of Parasitic Protozoa in Rio

02 April 2018: Our CRISPR Cas9 toolkit works also great in T. cruzi. Check out our latest publication (Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping) in collaboration with John Kelly's and Martin Taylor's laboratory.

29 November 2017: Our CRISPR Cas9 toolkit works also great in L. donovani. Check out our latest publication (Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit) in collaboration with Gerald Späth's laboratory.

12 September 2017: We have done a major update to our primer design tool and we hope that this version will be more user-friendly to access our repertoire of more than 120,000 primer sequences

12 September 2017: We have added Leishmania braziliensis MHOMBR75M2903 to our primer design tool and would like to thank Stephen Beverley for making this available to us

24 July 2017: Our published plasmid maps pTB011, pTB007, pTB008 and pRM006, as well as the common sgRNA primer sequence G00, are now also available for download on LeishGEdit

24 July 2017: We added Leishmania infantum JPCM5 to our primer design tool

24 July 2017: Check out our new pPLOT-Puro-eYFP-Puro plasmid for gene editing in Leishmania

03 May 2017: Our manuscript "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids" is now available for download. Get it here: Royal Society Open Science

03 April 2017: From today new tools on LeishGEdit are announced here!

29 March 2017: Our manuscript "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids" is now accepted for publication in Royal Society Open Science

27 March 2017: Primer design for Samuel Dean's pPOT plasmid are now available

27 March 2017: We added three more species, including TREU427, CL Brener and ParrotTarII, to our primer design tool

14 February 2017: Check out our new pPLOT plasmids for gene editing in Leishmania: pPLOT-Puro-mStrawberry-Puro, pPLOT-Puro-DD-Puro, pPLOT-Puro-TEV-mNeonGreen-Strep-Puro and pPLOT-Puro-DD-mNeonGreen-Puro

13 February 2017: Primer design is now easier to understand. We added more information on how to interpret the primer design output from LeishGEdit

16 December 2016: We added Leishmania donovani BPK282A1 to our primer design tool

21 October 2016: Four more species have been added to our primer design tool

Please contact Dr. Eva Gluenz (eva.gluenz@path.ox.ac.uk) or Tom Beneke (tobeneke@gmail.com) to request plasmids and for technical advice on primers, plasmids and cell lines. Please visit our lab website, if you have further interest in our research.

Citation

Please cite our main publication for using our website and method in any given organism: Beneke T., Madden R., Valli J., Makin L., Sunter J. and Gluenz E (2017). A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. Royal Society Open Science 10.1098/rsos.170095.

If you use our barcoding strategy or primer design with our latest CCTop pipeline please cite: Beneke and Gluenz, MBP 2020

Additionally we would like to ask you to cite Martel et al., BioMed Research International 2017 for publications in L. donovani and Costa et al., PLOS NTD 2018 for publications in T. cruzi when using our CRISPR Cas9 method.

Acknowledgements

We would like to thank Duo Peng (EuPaGDT) for giving access to a multithreaded batch mode to design sgRNAs, Samuel Dean for providing pPOTv4 plasmids and Andy Bassett for CRISPR advice. We also like to thank the authors of the CCTop toolbox, enabling us to design the latest LeishGEdit script version. The development of these tools was funded by the Wellcome Trust, the Medical Research Council (MRC), University of Oxford John Fell Fund and the Royal Society.

leishgedit

Website designed and maintained by Tom Beneke (tobeneke@gmail.com). Last updated on 5th August 2020.

LeishGEdit primer design

PLEASE NOTE: We recommend to use the new bar-seq primer design rather than the older version below!

With pPLOT / pT plasmids, which are optimised for gene expression in Leishmania spp., you can insert protein tags at the 5'- or 3'-end of a gene of interest or knock out a specific gene or locus in a kinetoplastid expressing Cas9 and T7 RNAP.

For knockouts and protein tagging in Trypanosoma spp. we recommend using pPOT plasmids (please contact Samuel Dean at the University of Warwick for plasmids1). Please note, pPOT plasmids are not optimised for use in Leishmania.

LeishGEdit has to date 122,170 primer sequences available for free. Please cite our website and publication (Beneke, Madden et al., 2017) so that we can continue this resource for the kinetoplastid community.

Please use the form below to retrieve your primer sequences for pT and pPLOT plasmids or pPOT plasmids. Although there is no input limit, we recommend that you use the Excel sheets below for queries larger than 1000 genes.

Design Primer Sequences

Please note that there are no pPOT primers available for Leishmania species as explained above.

Chose your desired gene editing strategy

N-terminal tagging

C-terminal tagging

Knockout

Tagging and knockout

Chose your desired modular plasmid system

pT and pPLOT plasmids

pPOT plasmids

You can export your designed primers as a '.csv' file on the search result page.

The common sgRNA scaffold primer is described in our publication (Beneke, Madden et al., 2017) and should be used for sgRNA template amplification. You can download the sequence here: G00.

Get a '.csv' file with a complete list of all primer sequences for gene editing with pT and pPLOT plasmids in one of the following organism:











Get CSV file with all primer sequences for gene editing with pPOT plasmids in one of the following organism:





How are primers designed?

The sgRNA primers contain the highest scoring 20nt guide RNA sequence the EuPaGDT CRISPR gRNA Design Tool identified within 105bp upstream or downstream of the target gene. Primers for amplification of the targeting cassettes contain 30nt of sequence immediately adjacent to the sgRNA target sequence. These are the 30nt homology arms for recombination. Shown below is a primer design example for N-terminal tagging of gene Tb927.11.7830.

Understanding the output for "Tagging and knockout"

"Upstream" and "Downstream" primers produced by LeishGEdit primer-design contain primer binding sites compatible with pT, pPLOT and pPOT plasmids; as well as 30nt homology arms for recombination. "sgRNA" primers consist of a T7 RNA polymerase promotor (for in vivo transcription of RNA), a 20nt sgRNA target sequence to indroduce the double strand break at a locus of interest and a 20nt overlap to the CRISPR-Cas9 backbone sequence allowing generation of sgRNA templates by PCR (follows the protocol published by Bassett and Liu, 2014).

Shown below on top is a primer design example for tagging and knockout primers of gene LdBPK_201450.1. with pT and pPLOT primers.

A primer design example for tagging and knockout primers of gene Tb927.11.11610 with pPOT primers is given on the bottom.

1 pPOT plasmids from Dean, S., Sunter, J., Wheeler, R., Hodkinson, I., Gluenz, E., Gull, K (2015). A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biology 5:140197.

2 Gene models from Fiebig, M., Kelly, S., Gluenz, E (2015). Comparative Life Cycle Transcriptomics Revises Leishmania mexicana Genome Annotation and Links a Chromosome Duplication with Parasitism of Vertebrates. PLoS Pathogens II:e1005186.

3 The genome of Leishmania braziliensis MHOM/BR/75/M2903 was sequenced by the the Genome Center at Washington University School of Medicine. Pre-publication access is a courtesy of Stephen M. Beverley.

Website designed and maintained by Tom Beneke (tobeneke@gmail.com). Last updated on 5th August 2020.

imgLeishGEdit bar-seq primer design

This is our new site for bar-seq primer design. We have modified our primer design tool for this purpose. Primers are now designed using a CCTop pipeline. To facilitate bar-seq assays using the LeishGEdit toolbox, the upstream forward primer (#1) is additionally barcoded as previously shown in Beneke et al., PLoS Pathogens 2019. The upstream forward primer is modified by inserting a 17 nt barcode and a 20 nt constant region in-between the existing 30 nt HF and 20 nt pT-pPLOT-pPOT primer binding site. Using the additional 20 nt constant region and 20 nt pT-pPLOT-pPOT primer binding site allows reading out barcode abundance by using Illumina amplicon sequencing strategies. The following cartoon gives an overview of this approach.

To see further details on how barcodes are generated and primers are designed using a CCTop pipeline please see our latest paper on MBP (Beneke and Gluenz, 2020).

Bar-seq primers produced by this script version are still compatible with pPLOT, pT and pPOT plasmids as described in previous versions. This latest version of LeishGEdit bar-seq primer design has currently 2,152,686 primer sequences for 39 different organisms available for free. Please cite our initial publication (Beneke, Madden et al., 2017), the LeishGEdit website and our latest MBP (Beneke and Gluenz, 2020) paper if you are using this website to design your bar-seq primers for bar-seq experiments so that we can continue to run this resource for the kinetoplastid community.

Please use the form below to retrieve your bar-seq primer sequences for pT and pPLOT plasmids or pPOT plasmids. Although there is no input limit, we recommend that you use the Excel sheets below for queries larger than 1000 genes.

imgDesign Bar-seq Primers (annotations based on TriTrypDB release 41)

You can export your designed bar-seq primer sequences as a '.csv' file on the search result page.

The common sgRNA scaffold primer is described in our publication (Beneke, Madden et al., 2017) and should be used for sgRNA template amplification. You can download the sequence here: G00.

Chose your desired gene editing strategy

N-terminal tagging

C-terminal tagging

Knockout

Tagging and knockout

Chose your desired modular plasmid system

pT and pPLOT plasmids

pPOT plasmids

Please note, when designing bar-seq primers for L. mexicana using gene models from Fiebig et al., PLoS Pathogens 2015 modify geneIDs to 'LmxMF' (instead of 'LmxM').

You can download a '.csv' file with a complete list of all bar-seq primer sequences for gene editing with pT, pPLOT and pPOT plasmids for any of the following organisms (annotations based on TriTrypDB release 41):

imgHow are bar-seq primers designed?

This new version of LeishGEdit primer design uses a CCTop (Stemmer et al., 2015) pipeline for the prediction of sgRNA target sites, and then designs donor DNA primers with sequences in close proximity to the sgRNA target sequence. The sgRNA target sequence is selected from a 130 nt search window upstream of the start codon or downstream of the stop codon. The highest scoring sgRNA within this window is chosen based on the CCTop scoring pipeline. The number of alignments to the genome with mismatches (MM) for any given sgRNA sequence is the main scoring criterion: Specifically, CCTop finds potential sgRNA target sites that have up to 2 MM in the first 12 nt upstream of the PAM site or up to 4 MM in the entire sgRNA target sequence and sorts these by least MM for the highest scoring sgRNA (as shown below).

Additionally, the number of perfect matches of the selected sgRNA sequence (23 nt, including the protospacer adjacent motif NGG) within the target genome is also determined. Since this is computed independently from the CCTop pipeline, this step also verifies the initial sgRNA design.

To provide an indication for off-targeting the bar-seq primer design outcome gives both these outputs: (1) the number of imperfect sgRNA matches with 1-4 MM in the target genome and (2) the number of perfect sgRNA matches in the target genome.

img Understanding the output for "Tagging and knockout"

"Upstream" and "Downstream" primers produced by LeishGEdit primer-design contain primer binding sites compatible with pT, pPLOT and pPOT plasmids; as well as 30nt homology arms for recombination. The upstream forward primer contains an additional p5 primer binding site and 17nt barcode to facilitate bar-seq screens using Illumina sequencing. "sgRNA" primers consist of a T7 RNA polymerase promotor (for in vivo transcription of RNA), a 20nt sgRNA target sequence to indroduce the double strand break at a locus of interest and a 20nt overlap to the sgRNA backbone sequence allowing generation of sgRNA templates by PCR (follows the protocol published by Bassett and Liu, 2014).

Shown below is a primer design example for tagging and knockout bar-seq primers of gene LdBPK_010110.1.1 with pT and pPLOT bar-seq primers and gene Tb927.5.5500 with pPOT bar-seq primers.

Website designed and maintained by Tom Beneke (tobeneke@gmail.com). Last updated on 5th August 2020.

Plasmids for gene editing

Plasmids below are available on request. You can find a detailed describtion of LeishGEdit in Beneke, Madden et al., 2017. For technical advice on primers, plasmids and cell lines please contact tobeneke@gmail.com or eva.gluenz@path.ox.ac.uk.

The common sgRNA primer G00 will not be shipped. Please order the G00 primer yourself.

Plasmid type Plasmids Version Resistance marker Fusion tag Amplicon size N-term Amplicon size C-term GeneBank file
pPLOT pPLOTv1 blast-mNeonGreen-blast 1 Blasticidin

Myc :: mNeonGreen (N-terminal)

mNeonGreen :: Myc (C-terminal)

2100bp 2500bp
pPLOTv1 neo-mNeonGreen-neo 1 Neomycin

Myc :: mNeonGreen (N-terminal)

mNeonGreen :: Myc (C-terminal)

2500bp 2900bp
pPLOTv1 puro-mNeonGreen-puro 1 Puromycin

Myc :: mNeonGreen (N-terminal)

mNeonGreen :: Myc (C-terminal)

2300bp 2700bp
pPLOTv1 phleo-mCherry-phleo 1 Phleomycin

Myc :: mCherry (N-terminal)

mCherry :: Myc (C-terminal)

2100bp 2500bp
pPLOTv1 puro-mCherry-puro 1 Puromycin

Myc :: mCherry (N-terminal)

mCherry :: Myc (C-terminal)

2300bp 2700bp
pPLOTv1 phleo-Halo-phleo 1 Phleomycin

Myc :: Ty :: Halo :: Ty (N-terminal)

Ty :: Halo :: Ty :: Myc (C-terminal)

2300bp 2700bp
pPLOTv1 puro-Halo-puro 1 Puromycin

Myc :: Ty :: Halo :: Ty (N-terminal)

Ty :: Halo :: Ty :: Myc (C-terminal)

2400bp 2800bp
pPLOTv1 puro-10Ty-puro 1 Puromycin

Myc :: 10Ty (N-terminal)

10xTy :: Myc (C-terminal)

2000bp 2300bp
pPLOTv1 puro-nanoLuc-puro 1 Puromycin

Myc :: Luciferase (N-terminal)

Luciferase :: Myc (C-terminal)

2150bp 2550bp
pPLOTv1 phleo-nanoLuc-phleo 1 Phleomycin

Myc :: Luciferase (N-terminal)

Luciferase :: Myc (C-terminal)

1900bp 2300bp
pPLOTv1 puro-mStrawberry-puro 1 Puromycin

Myc :: mStrawberry (N-terminal)

mStrawberry :: Myc (C-terminal)

2300bp 2700bp
pPLOTv1 puro-DD-puro 1 Puromycin

Myc :: Destabilization-domain (N-terminal)

Destabilization-domain :: Myc (C-terminal)

1900bp 2300bp
pPLOTv1 puro-DD-mNG-puro 1 Puromycin

Myc :: Destabilization-domain :: mNeonGreen (N-terminal)

Destabilization-domain :: mNeonGreen :: Myc (C-terminal)

2700bp 3000bp
pPLOTv1 puro-TEV-mNG-Strep-puro 1 Puromycin

Myc :: TEV :: mNeonGreen :: Strep (N-terminal)

TEV :: mNeonGreen :: Strep :: Myc (C-terminal)

2500bp 2900bp
pPLOTv1 puro-BirA*-puro 1 Puromycin

Myc :: BirA* (N-terminal)

BirA* :: Myc (C-terminal)

2600bp 3000bp
pPLOTv1 phleo-BirA*-phleo 1 Phleomycin

Myc :: BirA* (N-terminal)

BirA* :: Myc (C-terminal)

2400bp 2800bp
pPLOTv1 puro-eYFP-puro 1 Puromycin

Myc :: eYFP (N-terminal)

eYFP :: Myc (C-terminal)

2300bp 2700bp
pT

pTBlast_v1

1 Blasticidin 1700bp

pTNeo_v1

1 Neomycin 1750bp

pTPuro_v1

1 Puromycin 1800bp

pTB011

1 Blasticidin/Puromycin Flag::NLS::Cas9::NLS

pTB007

1 Hygromycin

Flag::NLS::Cas9::NLS

NLS::T7 RNAP

pTB008

1 Phleomycin

NLS::T7 RNAP

pRM006

1 Hygromycin

Flag::NLS::Cas9::NLS

G00 (is a primer sequence and not a plasmid)

1 common sgRNA primer for amplification

Technical notes:

pPLOT and pT serve as template DNA for PCR amplification of repair cassettes for genome editing by homologous recombination. The amplicons can be transfected together with the relevant sgRNA templates into a kinetoplastid cell line expressing Cas9 nuclease (for example derived from pX330 (Cong et al., 2013)) and T7 RNA polymerase for in vivo transcription of sgRNAs. Alternatively, the amplicons can be transfected together with in vitro transcribed sgRNAs into a kinetopastid cell line expressing Cas9 nuclease.

Generation of sgRNA templates by PCR follows the protocol published by Bassett and Liu, 2014. For complementation of knockout phenotypes we recommend integration of an add-back copy of the gene of interest into the beta tubulin locus.

Website designed and maintained by Tom Beneke (tobeneke@gmail.com). Last updated on 5th August 2020.