scholarly journals Robust and stable transcriptional repression in Giardia using CRISPRi

2018 ◽  
Author(s):  
SG McInally ◽  
KD Hagen ◽  
C Nosala ◽  
J Williams ◽  
K Nguyen ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Diarrheal Disease ◽  
Model Systems ◽  
Structural Defects ◽  
End Joining ◽  
Morpholino Knockdown ◽  
Endogenous Genes ◽  
Localization Signal ◽  
Ventral Disc ◽  
Non Homologous End Joining

AbstractGiardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of non-homologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disc MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than five days longer than transient morpholino-based knockdown. By expressing two gRNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disc defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows for rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

scholarly journals Robust and stable transcriptional repression in Giardia using CRISPRi

2019 ◽  
Vol 30 (1) ◽  
pp. 119-130 ◽  
Author(s):  
S. G. McInally ◽  
K. D. Hagen ◽  
C. Nosala ◽  
J. Williams ◽  
K. Nguyen ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Diarrheal Disease ◽  
Nonhomologous End Joining ◽  
Model Systems ◽  
Structural Defects ◽  
End Joining ◽  
Guide Rnas ◽  
Morpholino Knockdown ◽  
Endogenous Genes ◽  
Localization Signal

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

scholarly journals CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

2020 ◽  
Author(s):  
Yang Zhang ◽  
Jifeng Yuan
Keyword(s):  
Genome Editing ◽  
Transcriptional Repression ◽  
Gene Editing ◽  
Photosynthetic Bacteria ◽  
Genome Engineering ◽  
Biotechnological Applications ◽  
End Joining ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Non Homologous End Joining

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

scholarly journals Efficient replacement of long DNA fragments via non-homologous end joining at non-coding regions

2020 ◽  
Author(s):  
Shanye Gu ◽  
Jia Li ◽  
Siyuan Li ◽  
Jianbin Cao ◽  
Jiwen Bu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genomic Sequence ◽  
Genetic Manipulation ◽  
Expression Patterns ◽  
Egfp Expression ◽  
End Joining ◽  
Replacement Method ◽  
Endogenous Genes ◽  
Non Homologous End Joining ◽  
Genomic Regions

Abstract Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report a novel DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles of zebrafish. Functional assays proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. We further extended the application by making two EGFP reporter lines, in which the EGFP expression patterns were consistent with those of endogenous genes. Furthermore, combining double-fluorescence expression donor vectors, we showed that the DNA replacement efficiency of this NHEJ-mediated approach was around 3 times larger than that of HR/HDR-mediated approach. Together, we provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.

scholarly journals High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal

2020 ◽  
Vol 21 (11) ◽  
pp. 3747
Author(s):  
Jeong Pil Han ◽  
Yoo Jin Chang ◽  
Dong Woo Song ◽  
Beom Seok Choi ◽  
Ok Jae Koo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
High Efficiency ◽  
End Joining ◽  
Homology Directed Repair ◽  
Cas9 Protein ◽  
Localization Signal ◽  
Non Homologous End Joining ◽  
The Relationship ◽  
Donor Template

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

scholarly journals Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

2020 ◽  
Vol 9 ◽  
Author(s):  
Jerome Lacombe ◽  
Titouan Cretignier ◽  
Laetitia Meli ◽  
E. M. Kithsiri Wijeratne ◽  
Jean-Luc Veuthey ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Human Cancer ◽  
Repair Pathway ◽  
End Joining ◽  
Human Cancer Cells ◽  
Non Homologous End Joining

scholarly journals Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1506
Author(s):  
Angelos Papaspyropoulos ◽  
Nefeli Lagopati ◽  
Ioanna Mourkioti ◽  
Andriani Angelopoulou ◽  
Spyridon Kyriazis ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Mechanisms ◽  
Non Coding Rnas ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Living Organisms

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

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