Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania

Abstract

Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite Leishmania has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins.

Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

“…To overcome these limitations, there have been attempts to boost the CRISPR editing efficiency in Leishmania without requiring donor DNA. For example, Zhang et al (25) have recently attempted to reconstitute the NHEJ pathway of Mycobacterium marinum in Leishmania . While the authors claim that this improved DSB repair fidelity and efficiency in Leishmania , editing rates still remain low without the addition of donor DNA (less than 3% editing in selected non-clonal populations that express Cas9 and one sgRNA).…”

“…In consequence, CRISPR targeting in Leishmania is highly inefficient and usually requires extra steps to obtain the desired mutants. These extra steps typically involve either the addition of donor DNA (the mutation rate is less than 1% without donor DNA (7,8,25,31)) or other means, such as the selection of drug resistance-associated edits, or labour intensive cloning procedures (5,7,25). This makes pooled transfection loss-of-function screens currently unfeasible in Leishmania parasites.…”

“…For example, Cas9-expressing Toxoplasma gondii parasites can be transfected with sgRNA plasmid libraries to enable genome-wide CRISPR knockout screens (23). One of the reasons why similar pooled CRISPR transfection screens in Leishmania species have not been performed is that Leishmania parasites, unlike mammalian cells or T. gondii parasites, lack the key components of the non-homologous DNA end joining (NHEJ) pathway, including DNA-PKcs, Ligase IV, and XRCC4 (24,25). Following a Cas9introduced double-strand break (DSB), DNA in mammalian cells and T. gondii parasites is mainly repaired by NHEJ (>70% in humans, >90% in T. gondii of all DSBs), allowing indels and frame-shifts to be efficiently introduced in the target gene (26)(27)(28)(29)(30).…”

“…In contrast, Leishmania parasites rely on homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ) DNA repair pathways to repair Cas9-introduced DSBs. Without the addition of repair template DNA, this leads to large and unpredictable DNA deletions, increased cell death during DNA repair failures and prolonged repair times (7,8,25,31). In consequence, CRISPR targeting in Leishmania is highly inefficient and usually requires extra steps to obtain the desired mutants.…”

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

“…However, the applications of bar-seq screens are limited, and it remains difficult to target the vast Leishmania repertoire of repetitive genetic elements by gene replacement approaches. In addition, Leishmania parasites lack crucial components of the non-homologous DNA end joining (NHEJ) pathway (Passos-Silva et al, 2010; Zhang et al, 2022). As a result, CRISPR-induced DNA double-strand breaks (DSB) lead to unpredictable DNA deletions, increased cell death during DNA repair failures, prolonged repair times and generally low editing rates (Zhang et al, 2017; Zhang and Matlashewski, 2015; Zhang and Matlashewski, 2019; Zhang et al, 2022).…”