Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System

ABSTRACT Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. IMPORTANCE Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

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Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

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Staphylococcal DNA Repair Is Required for Infection

mBio ◽

10.1128/mbio.02288-20 ◽

2020 ◽

Vol 11 (6) ◽

Cited By ~ 2

Author(s):

Kam Pou Ha ◽

Rebecca S. Clarke ◽

Gyu-Lee Kim ◽

Jane L. Brittan ◽

Jessica E. Rowley ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Staphylococcus Aureus ◽

Human Blood ◽

Immune Cells ◽

Double Strand Breaks ◽

Oxygen Species ◽

Dna Double Strand Breaks ◽

Gram Positive ◽

Content Type ◽

Strand Breaks

ABSTRACT To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

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DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

Eukaryotic Cell ◽

10.1128/ec.00207-14 ◽

2015 ◽

Vol 14 (3) ◽

pp. 196-205 ◽

Cited By ~ 24

Author(s):

Bibo Li

Keyword(s):

Trypanosoma Brucei ◽

Surface Antigen ◽

Antigenic Variation ◽

Dna Recombination ◽

Double Strand Breaks ◽

Microbial Pathogens ◽

Dna Double Strand Breaks ◽

Content Type ◽

Strand Breaks ◽

Major Surface

ABSTRACTHuman-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation,Trypanosoma brucei(a kinetoplastid), which causes human African trypanosomiasis,Plasmodium falciparum(an apicomplexan), which causes malaria,Pneumocystis jirovecii(a fungus), which causes pneumonia, andBorrelia burgdorferi(a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except forPlasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed inT. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen inT. brucei. In subtelomeric VSG expression sites (ESs),VSGgenes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation inT. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review.

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Chlamydia trachomatis Inhibits Homologous Recombination Repair of DNA Breaks by Interfering with PP2A Signaling

mBio ◽

10.1128/mbio.01465-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 3

Author(s):

Yang Mi ◽

Rajendra Kumar Gurumurthy ◽

Piotr K. Zadora ◽

Thomas F. Meyer ◽

Cindrilla Chumduri

Keyword(s):

Cell Cycle ◽

Chlamydia Trachomatis ◽

Homologous Recombination ◽

Double Strand Breaks ◽

Host Cells ◽

Dna Double Strand Breaks ◽

Content Type ◽

Strand Breaks ◽

Recombination Repair ◽

Infected Cells

ABSTRACT Cervical and ovarian cancers exhibit characteristic mutational signatures that are reminiscent of mutational processes, including defective homologous recombination (HR) repair. How these mutational processes are initiated during carcinogenesis is largely unclear. Chlamydia trachomatis infections are epidemiologically associated with cervical and ovarian cancers. Previously, we showed that C. trachomatis induces DNA double-strand breaks (DSBs) but suppresses Ataxia-telangiectasia mutated (ATM) activation and cell cycle checkpoints. The mechanisms by which ATM regulation is modulated and its consequences for the repair pathway in C. trachomatis-infected cells remain unknown. Here, we found that Chlamydia bacteria interfere with the usual response of PP2A to DSBs. As a result, PP2A activity remains high, as the level of inhibitory phosphorylation at Y307 remains unchanged following C. trachomatis-induced DSBs. Protein-protein interaction analysis revealed that C. trachomatis facilitates persistent interactions of PP2A with ATM, thus suppressing ATM activation. This correlated with a remarkable lack of homologous recombination (HR) repair in C. trachomatis-infected cells. Chemical inhibition of PP2A activity in infected cells released ATM from PP2A, resulting in ATM phosphorylation. Activated ATM was then recruited to DSBs and initiated downstream signaling, including phosphorylation of MRE11 and NBS1 and checkpoint kinase 2 (Chk2)-mediated activation of the G2/M cell cycle checkpoint in C. trachomatis-infected cells. Further, PP2A inhibition led to the restoration of C. trachomatis-suppressed HR DNA repair function. Taking the data together, this study revealed that C. trachomatis modulates PP2A signaling to suppress ATM activation to prevent cell cycle arrest, thus contributing to a deficient high-fidelity HR pathway and a conducive environment for mutagenesis. IMPORTANCE Chlamydia trachomatis induces DNA double-strand breaks in host cells but simultaneously inhibits proper DNA damage response and repair mechanisms. This may render host cells prone to loss of genetic integrity and transformation. Here we show that C. trachomatis prevents activation of the key DNA damage response mediator ATM by preventing the release from PP2A, leading to a complete absence of homologous recombination repair in host cells.

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Delayed repletion of O6-methylguanine—DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity

Journal of Neurosurgery ◽

10.3171/jns.2003.98.3.0591 ◽

2003 ◽

Vol 98 (3) ◽

pp. 591-598 ◽

Cited By ~ 24

Author(s):

Yuichi Hirose ◽

Emiko L. Kreklau ◽

Leonard C. Erickson ◽

Mitchel S. Berger ◽

Russell O. Pieper

Keyword(s):

Cell Death ◽

Dna Methyltransferase ◽

Glioma Cells ◽

Double Strand Breaks ◽

Single Strand ◽

Dna Double Strand Breaks ◽

Content Type ◽

Strand Breaks ◽

Single Strand Breaks ◽

Fine Print

Object. Temozolomide (TMZ)-induced O6-methylguanine (MG) DNA lesions, if not removed by MG—DNA methyltransferase (MGMT), mispair with thymine, trigger rounds of futile mismatch repair (MMR), and in glioma cells lead to prolonged G2—M arrest and ultimately cell death. Depletion of MGMT by O6-benzylguanine (BG) sensitizes tumor cells to TMZ, and this combination is currently used in clinical trials. The use of the TMZ+BG combination in gliomas, however, is complicated by the prolonged TMZ-induced G2—M arrest, which may delay activation of poorly defined cell death pathways and allow for MGMT repletion and reversal of toxicity. Methods. To address these issues, the actions of TMZ were monitored in DNA MMR-proficient SF767 glioma cells depleted of MGMT by BG, and in cells in which BG was removed at various times after TMZ exposure. In MGMT-depleted cells, TMZ exposure led to DNA single-strand breaks and phosphorylation of cdc2, followed by G2—M arrest, induction of p53/p21, and DNA double-strand breaks. Although DNA single-strand breaks, phosphorylation of cdc2, and G2—M arrest could be reversed by repletion of MGMT up to 5 days after TMZ exposure, TMZ-induced cytotoxicity could only be prevented if MGMT was replenished within 24 hours of the onset of G2—M arrest, and before the creation of DNA double-strand breaks. Conclusions. These results indicate that although SF767 glioma cells undergo a prolonged G2—M arrest in response to TMZ, their ability to escape TMZ-induced cytotoxicity by MGMT repletion is limited to an approximately 24-hour period after the onset of G2—M arrest.

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Helicobacter pylori Infection Introduces DNA Double-Strand Breaks in Host Cells

Infection and Immunity ◽

10.1128/iai.02368-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4182-4189 ◽

Cited By ~ 52

Author(s):

Katsuhiro Hanada ◽

Tomohisa Uchida ◽

Yoshiyuki Tsukamoto ◽

Masahide Watada ◽

Nahomi Yamaguchi ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Genomic Instability ◽

Double Strand Breaks ◽

Host Cells ◽

Dna Double Strand Breaks ◽

Content Type ◽

Strand Breaks ◽

H Pylori

ABSTRACTGastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogenHelicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data thatH. pyloriitself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms ofH. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening ofH. pylori-infected human gastric biopsy specimens to identify candidate genes involved inH. pylori-induced host genomic instabilities. We found upregulation ofATMexpressionin vivoin gastric mucosal cells infected withH. pylori. Using gastric cancer cell lines, we confirmed that theH. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with bothcagpathogenicity island (PAI)-positive and -negative strains, but the effect was more robust withcagPAI-positive strains. These results are consistent with the fact that infections with bothcagPAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected withcagPAI-positive strains.

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Repair pathway choice for double-strand breaks

Essays in Biochemistry ◽

10.1042/ebc20200007 ◽

2020 ◽

Vol 64 (5) ◽

pp. 765-777 ◽

Cited By ~ 2

Author(s):

Yixi Xu ◽

Dongyi Xu

Keyword(s):

Cell Cycle ◽

Double Strand Breaks ◽

Homologous Region ◽

Gene Repair ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Environmental Radiation ◽

Strand Breaks ◽

Dna End Resection ◽

End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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