What is LeishGEdit?

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

imgWhat is new on LeishGEdit?

Contact

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 (tom.beneke@path.ox.ac.uk) 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

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.

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. 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 (tom.beneke@path.ox.ac.uk)

LeishGEdit primer design

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 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.

Primer design

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 CSV file with 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 (tom.beneke@path.ox.ac.uk)

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 tom.beneke@path.ox.ac.uk or eva.gluenz@path.ox.ac.uk.

Please note, due to the high number of requests, we are only shipping plasmids every first and third Monday of a month. 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 (tom.beneke@path.ox.ac.uk)