LeishBASEedit Primer Design

LeishBASEedit is a primer design tool to introduce STOP codons into any given gene of interest (based on TriTrypDB release 59). Our toolbox, described in Engstler and Beneke, eLife 2023, uses the hyBE4max cytosine base editor from the Li lab (Zhang et al., Nature Cell Biology 2020). This optimised cytosine base editor, has an expanded cytosine editing window within the single-guide RNA target sequence (editing window is between nucleotide position 4 and 12). To employ hyBE4max in Leishmania, the editor was cloned into pLdCH (Zhang et al., mSphere 2017). We named the resulting plasmid pLdCH-hyBE4max (see plasmid tab for a plasmid map).

In the search window below, users can design oligos that are needed to clone a single-guide RNA into pLdCH-hyBE4max or similar plasmids. Guide RNAs are designed through a CRISPR-CBEI pipeline (Yu et al., mSystems 2020). Below are detailed explanations of how this primer design pipeline works and how to interpret the primer design output.

At present, there are a total of 11,457,418 primer sequences available for 64 different species (based on TriTrypDB release 59). Please cite our website and publication (Engstler and Beneke, eLife 2023) so that we can continue this resource for the kinetoplastid community.

A protocol for cloning the guides into pLdCH-hyBE4max can be downloaded here:

A protocol for transfecting plasmids into Leishmania parasites can be downloaded here:

Please use the form below to retrieve single-guide RNAs and primer sequences for your gene of interest. Please use the downloadable '.csv' files for larger querries.

Design Primer Sequences

How are LeishBASEedit primers and guides designed?

Cytosine base editors are comprised of a cytidine deaminase domain (e.g. APOBEC-1) fused to an impaired form of Cas9 (e.g. D10A nickase) and to two monomers of uracil glycosylase inhibitors (UGI). Cytosine base editors do not require donor DNA or the introduction of double-strand breaks for gene editing. The deaminase domain facilitates a deamination, which transforms cytidine to uridine. Uridine is then repaired to thymidine during DNA replication or other DNA repair processes.

Cytosine base editors have specific editing windows within the single-guide RNA sequence, in-which cytidine to thymidine editing is efficient. In case of the hyBE4max editor from Zhang et al., Nature Cell Biology 2020, the editing window is at position 4-12 of the guide RNA target sequence (see figure below). There are four codons that result in a STOP codon, when cytidine is edited to thymidine (see figure below). LeishBASEedit designs single-guide RNAs that catalyse the editing of cytidine to thymidine in this 4-12 editing window and filters out only guides that target one of these four codons to transform them into a STOP codon. Guides are then filtered for targeting only the first 50% of the CDS. In addition, they are sorted based on their exact edit window (e.g. 4-8 has higher activity than 8-12) and based on how many STOP codons they would introduce. The assumption is that (in most cases) a complete knockout can be achieved by introducing a STOP codon within the first half of any given gene.

Shown below is a schematic of the hyBE4max editor and how it can be used to introduce STOP codons. For a detailed description please read our publication: Engstler and Beneke, eLife 2023.

Understanding the LeishBASEedit primer output

Upon submission of a gene ID into the search window above, LeishBASEedit will retrieve detailed information for all possible single-guide RNAs for the queried gene, as well as two primers that can be annealed and then cloned into pLdCH-hyBE4max (you can download the cloning protocol above). The figure below shows a primer design example for one of the guides in the PF16 gene (LmxM.20.1400). The left panel shows the cloning scheme that can be followed with the retrieved primers from LeishBASEedit. The panel on the right explains how to interpret the information provided by LeishBASEedit for each guide target sequence (following CBEI definitions: Yu et al., mSystems 2020).

Other LeishBASEedit primer output parameters

In addition to the 'Guide Target' output and the provided primers 1 and 2, LeishBASEedit prints out information for each guide designed for the gene of interest. An explanation for each of those parameters is provided in the table below.

LeishBASEedit output	Description
Gene ID	Gene ID querry by user
Guide ID	Unique guide ID assigned by LeishBASEedit
Strand	DNA strand where C to T editing takes place
CDS Position [%]	Percentage of the CDS that would remain if STOP codon is introduced
Guide Target	Guide target schematic as explained in figure above (panel on the right)
Guide Sequence	20 nt single-guide RNA sequence
Guide Coordinates	Coordinates of single-guide RNA sequence (start from 'ATG' of given gene)
Stop Codon Coordinates	Coordinates of newly introduced STOP codons (start from 'ATG' of given gene)
Editing Window	Coordinates of editing window (start from 'ATG' of given gene)
PAM Sequence	PAM sequence of given single-guide RNA
PAM Position	Coordinates of PAM sequence (start from 'ATG' of given gene)
Edited Sequence	Proceeding nucleotide and edited nucleotide of new STOP codon
Additional Stops	One scoring point per additionally introduced STOP codon will be given
Edit Window 4-8	Scoring point if C to T edit is within 4-8 window (guides with 4-8 edit window will have 2 points in total)
Edit Window 4-10	Scoring point if C to T edit is within 4-10 window (guides with 4-10 edit window will have 1 point in total)
20% CDS	Scoring point if C to T edit is within first 20% of CDS (guides within first 20% of CDS will have 2 points in total)
40% CDS	Scoring point if C to T edit is within first 40% of CDS (guides within first 40% of CDS will have 1 point in total)
Total score	Sum of guide score (used for guide ranking, if equal, guide closest to START codon used for ranking)
Perfect match count	Count of the perfect match of guide target sequence and its adjacent PAM site in the target genome
Primer 1 (guide reverse)	First of the two primers needed to clone the guide into pLdCH-hyBE4max (sequence needs no further modification for primer order)
Primer 2 (guide forward)	Second of the two primers needed to clone the guide into pLdCH-hyBE4max (sequence needs no further modification for primer order)

Website designed and maintained by Tom Beneke (tobeneke@gmail.com or tom.beneke@uni-wuerzburg.de). Last updated on 8th December 2022.

Plasmids for gene editing

Plasmids below can be used for base editing in Leishmania. A detailed description of each plasmid can be found in Engstler and Beneke, eLife 2023. Please contact Tom Beneke (tobeneke@gmail.com or tom.beneke@uni-wuerzburg.de) for plasmid requests, as well as for technical advice on primers, plasmids and cell lines.

Plasmid Name	Promoter	Expressed Protein	Resistance Marker	Size	GeneBank file
pLdCH-hyBE4max	L. donovani rRNA promoter	hyBE4max	Hygromycin	11,160 bp
pLdCH-hyBE4max-LmajDBD	L. donovani rRNA promoter	hyBE4max with Rad51 ssDNA binding domain (DBD) from L. major Friedlin	Hygromycin	11,271 bp
pTB007-hyBE4max	Intergenic driven/T7 RNAP	hyBE4max and T7 RNAP	Hygromycin	17,171 bp

Technical notes:

Below users can download protocols for guide cloning and transfection of plasmids into Leishmania parasites. Please cite Engstler and Beneke, eLife 2023 when using these protocol.

A protocol for cloning the guides into pLdCH-hyBE4max can be downloaded here:

A protocol for transfecting plasmids into Leishmania parasites can be downloaded here:

Website designed and maintained by Tom Beneke (tobeneke@gmail.com or tom.beneke@uni-wuerzburg.de). Last updated on 8th December 2022.