Conditional knockout of RAD51-related genes inLeishmania majorreveals 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 genomewide 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, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 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.

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

“…Third, both single and multigene families can be targeted with this system [ 16 , 30 ], and it even allows simultaneous editing of multiple loci [ 24 , 30 ], as well as the identification of essential genes [ 20 , 30 , 31 ]. CRISPR gene editing also allows for in situ addition of flanking loxP sites to a gene of interest and the subsequent rapamycin-inducible gene deletion by dimerisable Cre (DiCre) recombinase [ 32 , 33 ]. This facilitates deletion of essential genes and observation of the cell biological and morphological effects on living cells in a time-dependent manner.…”