LeishBASEedit Primer Design v2

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 and Herrmann May et al., BioRxiv 2024, 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 use the hyBE4max CBE in Leishmania, we have generated multiple CBE expression plasmids. Guides for base editing can be cloned into plasmids containing a ribosomal promoter or a modified T7 RNAP promoter (T7 T10 GG variant) for sgRNA expression. You can view these under the plasmid tab. As shown below, these guide expression constructs can be highly efficiently delivered and integrated into the Leishmania genome using AsCas12a (for a detailed method description see Herrmann May et al., BioRxiv 2024).

In the search window below, users can design oligos that are needed to clone a single-guide RNA into guide expression plasmids. Guide RNAs are designed through a CRISPR-CBEI pipeline (Yu et al., mSystems 2020). Below is a detailed explanation 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 and Herrmann May et al., BioRxiv 2024) so that we can continue this resource for the kinetoplastid community.

A protocol for cloning the guides into guide expression plasmids can be downloaded here:

A protocol for using Cas12a for stable integration of CBE guide expression constructs into Leishmania parasites 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. The guide ranking is based on our updated ranking tool (LeishBASEedit v2: Herrmann May et al., BioRxiv 2024). 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. The assumption is that (in most cases) a functional mutation can be achieved by introducing a STOP codon within the first half of any given gene. In addition, guide ranking is also affected by the position of the STOP codon within the editing window (e.g. 4-8 has higher activity than 8-12) and by the number of possible STOP codons that could be introduced. Lastly, guides are ranked to have a low cytidine to STOP codon ratio. This allows to prioritize CBE sgRNAs that ensure STOP codon generation and eliminates competition between deleterious and non-deleterious mutations. You can find more infos about guide scoring in our recent toolbox release (LeishBASEedit v2: Herrmann May et al., BioRxiv 2024).

Shown below is a schematic of the hyBE4max base editor and how it can be used to introduce STOP codons. For a detailed description please read our publications: Engstler and Beneke, eLife 2023 and (LeishBASEedit v2: Herrmann May et al., BioRxiv 2024).

Understanding the LeishBASEedit primer output

Upon submission of a gene ID into the search window above, LeishBASEedit v2 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 a sgRNA expression plasmid (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 (top left panel) and the guide scoring/ranking scheme as described in Herrmann May et al., BioRxiv 2024(bottom left panel). Primers are provided for cloning into rRNA and T7 T10 GG promoter-driven sgRNA expression vectors. 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 v2	Unique guide ID assigned by LeishBASEedit v2
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
Perfect match count	Count of the perfect match of guide target sequence and its adjacent PAM site in the target genome
Number of Cytosines	Number of cytosines within the 4-12nt editing window
Number of possible STOP codons	Number of possible STOP codons when C is converted to T within the 4-12nt editing window
Ratio C to STOP	Number of cytosines divided by number of possible STOP codons (both within the 4-12nt editing window)
Edit Window 4-8	Checks whether the C to T edit is within the 4-8 editing window
Edit Window 4-10	Checks whether the C to T edit is within the 4-10 editing window
C score	Guide score (see Herrmann May et al., BioRxiv 2024 for more info)
Primer 1 (guide reverse)	First of the two primers needed to clone the guide into sgRNA expression vectors (sequence needs no further modification for primer order)
Primer 2 (rRNAP guide forward)	Second of the two primers needed to clone the guide into rRNA promoter sgRNA expression vectors (sequence needs no further modification for primer order)
Primer 2 (T7 T10 GG guide forward)	First of the two primers needed to clone the guide into T7 T10 GG promoter sgRNA expression vectors (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 28th February 2024.

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 and Herrmann May et al., BioRxiv 2024. 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	Plasmid purpose	Resistance Marker	Size	GeneBank file
pLdCH-hyBE4max	L. donovani rRNA promoter	Co-expression of sgRNA and hyBE4max	Hygromycin	11,160 bp
pLdCH-hyBE4max-LmajDBD	L. donovani rRNA promoter	Co-expression of sgRNA and hyBE4max with Rad51 ssDNA binding domain (DBD) from L. major Friedlin	Hygromycin	11,271 bp
pTB007-hyBE4max	Intergenic driven	Co-expression of hyBE4max and T7 RNAP	Hygromycin	17,171 bp
pTB106	Intergenic driven	Co-expression of hyBE4max-LmajDBD, T7 RNAP and AsCas12a ultra (M537R/F870L mutation)	Hygromycin	21,608 bp
pTB107	Intergenic driven	Co-expression of hyBE4max, T7 RNAP and AsCas12a ultra (M537R/F870L mutation)	Hygromycin	21,498 bp
pTB102	T7 T10 GG promoter variant	Expression of sgRNA; plasmid without homology arms for integration	Puromycin	3,849 bp
pTB104	T7 T10 GG promoter variant	Expression of sgRNA; plasmid with homology arms for integration into NeoR gene	Puromycin	4,557 bp
pTB105	T7 T10 GG promoter variant	Expression of sgRNA; plasmid with homology arms for integration into 18S rRNA SSU locus	Puromycin	4,491 bp

Technical notes:

Below users can download protocols for guide cloning, Cas12a-mediated integration of expression constructs and transfection of DNA constructs into Leishmania parasites. Please cite Engstler and Beneke, eLife 2023 and Herrmann May et al., BioRxiv 2024 when using these protocol.

A protocol for cloning the guides into guide expression plasmids can be downloaded here:

A protocol for using Cas12a for stable integration of CBE guide expression constructs into Leishmania parasites 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 28th February 2024.