Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Abstract

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase

“…Consistent with the data shown by Beneke et al. (2017) and Damasceno and Reis-Cunha (2020) in L. major stably expressing Cas9 and T7, no significant differences in parasite growth in culture were observed when the Lb C9/T7 cell line was compared with the wild-type strain (Lb WT). Additionally, no differences in metacyclogenesis rates and in infectivity were observed in Lb C9/T7 when compared with Lb WT showing that the stable expression of Cas9 and T7 RNA polymerase does not cause fitness loss in L. braziliensis .…”

“…CRISPR/Cas9 has facilitated genome editing by allowing complete gene removal in one round of transfection, with no cloning steps. The strategy has already been used to unveil gene function in different Leishmania species (Beneke et al, 2017;Martel et al, 2017;Grewal et al, 2019;Shrivastava et al, 2019;Tupperwar et al, 2019;Adaui et al, 2020;Burge and Damianou, 2020;Damasceno and Reis-Cunha, 2020;Damianou et al, 2020;Espada et al, 2021;Turra et al, 2021) so far, been made in the species of the Leishmania subgenus and using episomal expression of Cas9. Recently, Adaui et al (2020) published the first genome editing using CRISPR/Cas9 in L. braziliensis.…”

“…Generation of sgRNA templates and donor DNAs is accomplished by PCR using primers retrieved by the LeishGEdit platform upon Gene ID input ( Beneke and Gluenz, 2019 ). Using this system, genes were successfully deleted in a variety of Leishmania species including Leishmania mexicana ( Beneke et al., 2017 ; Grewal et al., 2019 ; Shrivastava et al., 2019 ; Tupperwar et al., 2019 ; Burge and Damianou, 2020 ; Damianou et al., 2020 ), L. major ( Beneke et al., 2017 ; Damasceno and Reis-Cunha, 2020 ; Espada et al., 2021 ), Leishmania tarentolae ( Turra et al., 2021 ), L. donovani ( Martel et al., 2017 ), and L. braziliensis ( Adaui et al., 2020 ). Except for L. major ( Beneke et al., 2017 ; Espada et al., 2021 ), in all other cases, Cas9 and T7 RNA polymerase were expressed via an episome, requiring the maintenance of drug selection to avoid plasmid loss.…”

Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets

“…Unfortunately, inducible systems to turn gene expression on and off in Leishmania have not been explored, and there is limited literature regarding the use of these systems ( Yan et al., 2001 ; Kushnir et al., 2005 ; Yao et al., 2007 ; Kraeva et al., 2014 ). Although the implementation of the DiCRE system offers significant advancement in this regard, there are still certain limitations because it is neither reversible nor tunable ( Duncan et al., 2016 ; Santos et al., 2017 ; Damasceno et al., 2020b ; Damianou et al., 2020 ).…”

Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review

“…The adaptation and optimization of this approach for Leishmania spp. led to genetic engineering, identification of essential genes, and characterization of potential drug targets [125,126]. In particular, the method was improved in a CRISPR-Cas9 toolkit for quick and precise gene modification by integration of donor DNA, using engineered cell lines and drug selection of mutants, which was developed in L. major, L. mexicana [127] and validated in L. donovani [128].…”