Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Abstract

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

Improved base editing and functional screening inLeishmaniavia co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase, and a cytosine base editor

“…While our usual design for CBE sgRNAs aims to introduce STOP codons through cytosine-to-thymine conversion, we selected PF16-3 for this test because its editing activities are known to vary across species (Engstler and Beneke, 2023). In earlier attempts, we had to express CBE sgRNA PF16-3 for 42 days to achieve sufficient editing in L. major when using our pLdCH-hyBE4max single vector (Figure 1A) (Engstler and Beneke, 2023). Meanwhile, in L. donovani, high editing rates were already achieved 14 days post transfection.…”

“…shown to increase editing activity (Gier et al, 2020). We then amplified the synthesized Cas12a construct in a fusion PCR, adding parts of the T7 RNAP (ORF) and an intergenic region to the construct (using primer-pair 2051F/2052R and 2053F/2054R for amplification from pTB007-hyBE4max).…”

“…To test the Cas12a-mediated integration efficiency of donor DNA constructs with varying homology flank lengths, we generated a DNA construct containing mNeonGreen (mNG) as a reporter and blasticidin as a resistance marker, as well as ~600 nt homology flanks targeting the 18S rRNA SSU locus (Misslitz et al, 2000;Sorensen et al, 2003). This construct was assembled by fusion PCR as previously described (Engstler and Beneke, 2023), using pPLOT-mNG-Blast (Beneke et al, 2017) as a PCR template. The generated fusion construct was then cloned into a blunted vector backbone, resulting in the plasmid pPLOT-SSU-HDR-mNG-Blast.…”

“…Cells were transfected using transfection cuvettes supplied with the Basic Parasite Nucleofector Kit (Lonza transfection) or 2 mm BTX cuvettes (Tb-BSF transfection) with one pulse of the X-001 program on an Amaxa Nucleofector 2b (Lonza). For measuring transfection efficiencies, cells were selected and diluted on 96-well plates as described (Engstler and Beneke, 2023).…”

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites

“…Engstler & Beneke, 2023). It appears feasible to adapt CBE to splitCas9 to establish a conditional gene editing system in apicomplexan parasites.While the use of indicator strains allows for the screening of specific pathways and mechanisms, for example for genes involved in organelle biosynthesis, cytoskeletal dynamics, host cell motility or signalling (for an overview of potential indicators see Figure 2), especially screens for parasites blocked in natural F I G U R E 2 Versatility of splitCas9 screens.…”