scholarly journals A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica

2021 ◽  
Vol 87 (6) ◽  
Author(s):  
Zhiyong Cui ◽  
Huihui Zheng ◽  
Jinhong Zhang ◽  
Zhennan Jiang ◽  
Ziwei Zhu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Phase Synchronization ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Content Type ◽  
Genome Integration ◽  
Targeted Integration ◽  
Integration Sites ◽  
Iterative Integration

ABSTRACT Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica. Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica. Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolytica. IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.

scholarly journals Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joana Ferreira da Silva ◽  
Sejla Salic ◽  
Marc Wiedner ◽  
Paul Datlinger ◽  
Patrick Essletzbichler ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Genetic Ablation ◽  
Alternative End Joining ◽  
Non Homologous End Joining ◽  
Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

scholarly journals A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei-Yi Yan ◽  
Si-Shang Li ◽  
Xin-Yuan Ding ◽  
Xiao-Peng Guo ◽  
Qi Jin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Editing ◽  
Large Scale ◽  
Nonhomologous End Joining ◽  
Genetic Mutations ◽  
Repair Pathway ◽  
End Joining ◽  
Content Type ◽  
Nhej Repair ◽  
Short Palindromic Repeat

ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

scholarly journals Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

2015 ◽  
Vol 35 (14) ◽  
pp. 2448-2463 ◽  
Author(s):  
Matthew Jessulat ◽  
Ramy H. Malty ◽  
Diem-Hang Nguyen-Tran ◽  
Viktor Deineko ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Large Scale ◽  
Spindle Assembly Checkpoint ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Cell Cycle Regulators ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Content Type

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.

scholarly journals Precision genome editing in the CRISPR era

2017 ◽  
Vol 95 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Jayme Salsman ◽  
Graham Dellaire
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Point Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genetic Changes ◽  
New Era ◽  
Homology Directed Repair ◽  
Repair Template ◽  
Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

scholarly journals Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase

2015 ◽  
Vol 81 (13) ◽  
pp. 4423-4431 ◽  
Author(s):  
Tao Xu ◽  
Yongchao Li ◽  
Zhou Shi ◽  
Christopher L. Hemme ◽  
Yuan Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Step ◽  
Seed Region ◽  
End Joining ◽  
Content Type ◽  
Clostridium Cellulolyticum ◽  
Interval Distance ◽  
Gene Coverage ◽  
One Step ◽  
Foreign Genes

ABSTRACTThe CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of theStreptococcus pyogenesCRISPR-Cas9 system in editing the genome ofClostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal toC. cellulolyticumdue to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage inC. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.

scholarly journals Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yinghang Liu ◽  
Xin Jiang ◽  
Zhiyong Cui ◽  
Zhaoxuan Wang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Mevalonate Pathway ◽  
Lipid Production ◽  
Economic Value ◽  
Culture Conditions ◽  
End Joining ◽  
Expression Library ◽  
Cofactor Specificity ◽  
Non Homologous End Joining ◽  
Iterative Integration

Abstract Background Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. Results We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. Conclusions Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

scholarly journals CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Greg Matlashewski
Keyword(s):  
Visceral Leishmaniasis ◽  
Genome Editing ◽  
Cleavage Site ◽  
Leishmania Donovani ◽  
Expression Vectors ◽  
Drug Selection ◽  
End Joining ◽  
Content Type ◽  
Homology Directed Repair ◽  
Cas9 Nuclease

ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. IMPORTANCE Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

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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