Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania

Abstract

CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania. Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania. In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in Leishmania. IMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

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

“…The most active sgRNAs (sets 5 and 6) were the same in L. braziliensis and L. donovani , indicating that the sgRNA sequence had an impact on the gene targeting efficiency. This is in line with a recent study that tested the efficiency of three gRNAs targeting identical sequences of the miltefosine transporter gene in L. donovani , L. major , and L. mexicana , and found the relative gRNA activity to be the same [ 31 ]. Studies in other systems revealed that sgRNA sequence features such as position-specific nucleotide composition, GC content, motifs located in the sgRNA “seed” region, and secondary structures of sgRNAs contribute to sgRNA efficacy [ 80 , 81 , 82 , 83 , 84 ].…”

“…Second, the generation of CRISPR-derived null mutants is facilitated by the use of donor DNA repair cassettes (containing antibiotic selection markers) flanked by short homology arms targeting the gene of interest (GOI), in a single transfection [ 17 , 29 ]. 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 ].…”

“…In the absence of a donor DNA repair template, Leishmania use microhomology-mediated end-joining (MMEJ) or single-strand annealing (SSA) to repair DSBs, both of which lead to deletions of various sizes that disrupt the targeted gene [ 20 , 30 , 31 ]. These DSB repair pathways (MMEJ and SSA) have a generally low efficiency in Leishmania , and SSA may result in unwanted deletions of adjacent genes [ 31 ].…”

“…In the absence of a donor DNA repair template, Leishmania use microhomology-mediated end-joining (MMEJ) or single-strand annealing (SSA) to repair DSBs, both of which lead to deletions of various sizes that disrupt the targeted gene [ 20 , 30 , 31 ]. These DSB repair pathways (MMEJ and SSA) have a generally low efficiency in Leishmania , and SSA may result in unwanted deletions of adjacent genes [ 31 ]. Transfections of a donor DNA template to facilitate homology-directed repair significantly improves CRISPR–Cas9 gene targeting efficiency and specificity, and eases the identification of CRISPR-edited mutants in Leishmania [ 17 , 19 , 20 , 30 , 31 ].…”

“…These DSB repair pathways (MMEJ and SSA) have a generally low efficiency in Leishmania , and SSA may result in unwanted deletions of adjacent genes [ 31 ]. Transfections of a donor DNA template to facilitate homology-directed repair significantly improves CRISPR–Cas9 gene targeting efficiency and specificity, and eases the identification of CRISPR-edited mutants in Leishmania [ 17 , 19 , 20 , 30 , 31 ].…”