scholarly journals Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

2021 ◽  
Author(s):  
Soujanya Akella ◽  
Xinrong Ma ◽  
Romana Bacova ◽  
Zachary P Harmer ◽  
Martina Kolackova ◽  
...  
Keyword(s):  
Target Genes ◽  
Selectable Marker ◽  
Marker Gene ◽  
Acetolactate Synthase ◽  
Gene Repair ◽  
Dna Breaks ◽  
Tetratricopeptide Repeats ◽  
Cellular Processes ◽  
Homology Directed Repair ◽  
Double Stranded Dna

Abstract Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 (WDTC1) genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.

scholarly journals Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

2021 ◽  
Author(s):  
Heriberto D. Cerutti ◽  
Soujanya Akella ◽  
Xinrong Ma ◽  
Romana Bacova ◽  
Zachary Harmer ◽  
...  
Keyword(s):  
Target Genes ◽  
Selectable Marker ◽  
Marker Gene ◽  
Acetolactate Synthase ◽  
Gene Repair ◽  
Dna Breaks ◽  
Cellular Processes ◽  
Homology Directed Repair ◽  
Double Stranded Dna ◽  
Targeting Strategy

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

2009 ◽  
Vol 28 (5) ◽  
pp. 769-776 ◽  
Author(s):  
Makoto Tougou ◽  
Noriko Yamagishi ◽  
Noriyuki Furutani ◽  
Koichiro Kaku ◽  
Tsutomu Shimizu ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Marker Gene ◽  
Acetolactate Synthase ◽  
Selectable Marker Gene

scholarly journals The Origins and Consequences of Localized and Global Somatic Hypermutation

2018 ◽  
Author(s):  
Fouad Yousif ◽  
Stephenie D. Prokopec ◽  
Ren X. Sun ◽  
Fan Fan ◽  
Christopher M. Lalansingh ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Target Genes ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Dna Breaks ◽  
Specific Effects ◽  
Double Stranded Dna ◽  
Mutation Density ◽  
Cancer Types ◽  
Mutational Processes

AbstractCancer is a disease of the genome, but the dramatic inter-patient variability in mutation number is poorly understood. Tumours of the same type can differ by orders of magnitude in their mutation rate. To understand potential drivers and consequences of the underlying heterogeneity in mutation rate across tumours, we evaluated both local and global measures of mutation density: both single-stranded and double-stranded DNA breaks in 2,460 tumours of 38 cancer types. We find that SCNAs in thousands of genes are associated with elevated rates of point-mutations, while similarly point-mutation patterns in dozens of genes are associated with specific patterns of DNA double-stranded breaks. These candidate drivers of mutation density are enriched for known cancer drivers, and preferentially occur early in tumour evolution, appearing clonally in all cells of a tumour. To supplement this understanding of global mutation density, we developed and validated a tool called SeqKat to identify localized “rainstorms” of point-mutations (kataegis). We show that rates of kataegis differ by four orders of magnitude across tumour types, with malignant lymphomas showing the highest. Tumours with TP53 mutations were 2.6-times more likely to harbour a kataegic event than those without, and 239 SCNAs were associated with elevated rates of kataegis, including loss of the tumour-suppressor CDKN2A. We identify novel subtypes of kataegic events not associated with aberrant APOBEC activity, and find that these are localized to specific cellular regions, enriched for MYC-target genes. Kataegic events were associated with patient survival in some, but not all tumour types, highlighting a combination of global and tumour-type specific effects. Taken together, we reveal a landscape of genes driving localized and tumour-specific hyper-mutation, and reveal novel mutational processes at play in specific tumour types.

MiRNA-145 and Its Direct Downstream Targets in Digestive System Cancers: A Promising Therapeutic Target

2020 ◽  
Vol 26 ◽  
Author(s):  
Yini Ma ◽  
Xiu Cao ◽  
Guojuan Shi ◽  
Tianlu Shi
Keyword(s):  
Digestive System ◽  
Target Genes ◽  
Malignant Neoplasm ◽  
Vital Role ◽  
Diagnostic Biomarkers ◽  
Cellular Processes ◽  
Promising Therapeutic Target ◽  
Direct Target ◽  
Potential Strategy

: MicroRNAs (miRNAs) play a vital role in the onset and development of many diseases, including cancers. Emerging evidence shows that numerous miRNAs have the potential to be used as diagnostic biomarkers for cancers, and miRNA-based therapy may be a promising therapy for the treatment of malignant neoplasm. MicroRNA-145 (miR-145) has been considered to play certain roles in various cellular processes, such as proliferation, differentiation and apoptosis, via modulating expression of direct target genes. Recent reports show that miR-145 participates in the progression of digestive system cancers, and plays crucial and novel roles for cancer treatment. In this review, we summarize the recent knowledge concerning the function of miR-145 and its direct targets in digestive system cancers. We discuss the potential role of miR-145 as valuable biomarkers for digestive system cancers and how miR-145 regulates these digestive system cancers via different targets to explore the potential strategy of targeting miR-145.

scholarly journals Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 542
Author(s):  
Gustavo Penteado Battesini Carretero ◽  
Greice Kelle Viegas Saraiva ◽  
Magali Aparecida Rodrigues ◽  
Sumika Kiyota ◽  
Marcelo Porto Bemquerer ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Biological Activities ◽  
Biological Effects ◽  
Membrane Surface ◽  
Helical Structure ◽  
Helical Conformation ◽  
Property A ◽  
Cellular Processes ◽  
Double Stranded Dna ◽  
Low Toxicity

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

scholarly journals New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

2020 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
Jaiana Malabarba ◽  
Elisabeth Chevreau ◽  
Nicolas Dousset ◽  
Florian Veillet ◽  
Julie Moizan ◽  
...  
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Acetolactate Synthase ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Adventitious Regeneration ◽  
Base Editing ◽  
Perennial Plants ◽  
Guide Rnas ◽  
Albino Phenotype

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

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