Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>

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, homolog...

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Autores principales:	Wen-Wei Zhang, Greg Matlashewski
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR-Cas9 DNA polymerase theta Leishmania SaCas9 double-strand DNA break repair gene deletion Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6a04c6b29f2849d5825dd05b8dcbe129
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spelling	oai:doaj.org-article:6a04c6b29f2849d5825dd05b8dcbe1292021-11-15T15:22:27ZSingle-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>10.1128/mSphere.00408-192379-5042https://doaj.org/article/6a04c6b29f2849d5825dd05b8dcbe1292019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00408-19https://doaj.org/toc/2379-5042ABSTRACT 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.Wen-Wei ZhangGreg MatlashewskiAmerican Society for MicrobiologyarticleCRISPR-Cas9DNA polymerase thetaLeishmaniaSaCas9double-strand DNA break repairgene deletionMicrobiologyQR1-502ENmSphere, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas9 DNA polymerase theta Leishmania SaCas9 double-strand DNA break repair gene deletion Microbiology QR1-502
spellingShingle	CRISPR-Cas9 DNA polymerase theta Leishmania SaCas9 double-strand DNA break repair gene deletion Microbiology QR1-502 Wen-Wei Zhang Greg Matlashewski Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
description	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.
format	article
author	Wen-Wei Zhang Greg Matlashewski
author_facet	Wen-Wei Zhang Greg Matlashewski
author_sort	Wen-Wei Zhang
title	Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
title_short	Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
title_full	Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
title_fullStr	Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
title_full_unstemmed	Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>
title_sort	single-strand annealing plays a major role in double-strand dna break repair following crispr-cas9 cleavage in <italic toggle="yes">leishmania</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/6a04c6b29f2849d5825dd05b8dcbe129
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Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in <italic toggle="yes">Leishmania</italic>

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