Mycobacterium tuberculosisandMycobacterium marinumnon-homologous end-joining proteins can function together to join DNA ends inEscherichia coli

Wright, Douglas G.; Castore, Reneau; Shi, Runhua; Mallick, Amrita; Ennis, Don G.; Harrison, Louis

doi:10.1093/mutage/gew042

Cited by 8 publications

(13 citation statements)

References 47 publications

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“…brucei cells to treatment with DSB-inducing reagents (67). Since NHEJ is the most efficient DSBR pathway in mammalian cells, to improve DSBR efficiency in Leishmania and overcome the possible hindering effect of Ku70/80 on other DSBR pathways, it will be interesting to determine whether it is possible to reconstitute the NHEJ pathway in Leishmania by transgenic expression of ligase IV and XRCC4 from humans or other organisms, as in yeast and Escherichia coli, where the NHEJ pathway is reconstituted by the transgenic expression of mycobacterial Ku and ligase proteins (68, 69). In mammalian cells and yeast, the Rad52 protein plays a central role in HR and SSA, as it interacts directly with both RPA and Rad51 to promote the assembly of the Rad51 nucleoprotein on ssDNA and assist with the homology search and complementary ssDNA annealing (45).…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

Matlashewski

2019

mSphere

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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.

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Section: Discussionmentioning

confidence: 99%

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

Zhang

Matlashewski

2019

mSphere

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“…A recent study found that M. smegmatis Ku can bind to DNA without free ends and this property was attributed to its lysine-rich C-terminal extension ( 49 ). MmKu has a similar C-terminal extension ( 32 , 50 ).…”

Section: Discussionmentioning

confidence: 99%

“…Nuclear-targeted MtKu also attenuates homologous recombination in mammalian cells ( 31 ). Analogous to M. tuberculosis, M. marinum was recently shown to possess a proficient NHEJ system ( 32 ). We have determined that M. marinum Ku (MmKu) is expressed at a higher level than MtKu in bacterial ( 32 ) and mammalian cells (this study).…”

Section: Introductionmentioning

confidence: 99%

“…Analogous to M. tuberculosis, M. marinum was recently shown to possess a proficient NHEJ system ( 32 ). We have determined that M. marinum Ku (MmKu) is expressed at a higher level than MtKu in bacterial ( 32 ) and mammalian cells (this study). Hence, we selected mitochondrial-targeted MmKu as a molecular tool to bind to DSBs in ρ + mtDNA to test the hypothesis that DSB-bound MmKu could prevent mtDNA replication or repair.…”

Section: Introductionmentioning

confidence: 99%

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Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

Prasai

Robinson

Scott

et al. 2017

Nucleic Acids Research

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The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells.

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“…Combined with the CRISPR system, HDR provides accurate and markerless target gene deletion, mutation, and insertion of foreign DNA sequences (Cobb et al, 2015). Alternatively, in some bacterial species such as Mycobacterium smegmatis, DSB can be repaired by the NHEJ system, which comprises an ATP-dependent DNA ligase and a Ku protein (Wright et al, 2017;Zheng et al, 2017). Unlike HDR, the NHEJ system does not require homologous DNA sequences for recombination, but directly joins the breaks, facilitating convenient gene deletion and insertion (Babynin, 2007).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

Zhou

Liu

et al. 2020

Front. Bioeng. Biotechnol.

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Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.

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Mycobacterium tuberculosisandMycobacterium marinumnon-homologous end-joining proteins can function together to join DNA ends inEscherichia coli

Cited by 8 publications

References 47 publications

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

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

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

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