Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

Making targeted gene deletions is essential for studying organisms, but is difficult in many prokaryotes due to the inefficiency of homologous recombination based methods. Here, I describe an easily modifiable, single-plasmid system that can be used to make rapid, sequence targeted, markerless knockouts in both a Gram-negative and a Gram-positive organism. The system is comprised of targeted DNA cleavage by Cas9 and error-prone repair by Non-Homologous End Joining (NHEJ) proteins. I confirm previous results showing that Cas9 and NHEJ can make knockouts when NHEJ is expressed before Cas9. Then, I show that Cas9 and NHEJ can be used to make knockouts when expressed simultaneously. I term the new method Potentially Organism-Agnostic Knockout (POAK) system and characterize its function in Escherichia coli and Weissella confusa. First, I develop a novel transformation protocol for W. confusa. Next, I show that, as in E. coli, POAK can create knockouts in W. confusa. Characterization of knockout efficiency across galK in both E. coli and W. confusa showed that while all gRNAs are effective in E. coli, only some gRNAs are effective in W. confusa, and cut site position within a gene does not determine knockout efficiency for either organism. I examine the sequences of knockouts in both organisms and show that POAK produces similar edits in both E. coli and W. confusa. Finally, as an example of the importance of being able to make knockouts quickly, I target W. confusa sugar metabolism genes to show that two sugar importers are not necessary for metabolism of their respective sugars. Having demonstrated that simultaneous expression of Cas9 and NHEJ is sufficient for making knockouts in two minimally related bacteria, POAK represents a hopeful avenue for making knockouts in other under-utilized bacteria.

Double-strand DNA breaks recruit the centromeric histone CENP-A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908233106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15762-15767 ◽

Cited By ~ 90

Author(s):

Samantha G. Zeitlin ◽

Norman M. Baker ◽

Brian R. Chapados ◽

Evi Soutoglou ◽

Jean Y. J. Wang ◽

...

Keyword(s):

Dna Cleavage ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Strand Breaks ◽

Double Strand Dna Breaks ◽

Histone H3 Variant ◽

Radiation Induced ◽

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

Journal of Radiation Research ◽

10.1093/jrr/rraa059 ◽

2020 ◽

Vol 61 (5) ◽

pp. 639-647

Author(s):

Yan Du ◽

Yoshihiro Hase ◽

Katsuya Satoh ◽

Naoya Shikazono

Keyword(s):

Gamma Rays ◽

Wild Type ◽

End Joining ◽

Complex Type ◽

Single Base ◽

In The Wild ◽

Dna Ends ◽

Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

10.1101/2021.11.01.466786 ◽

2021 ◽

Author(s):

Eleonora I. Ioannidi ◽

Matthew T. N. Yarnall ◽

Cian Schmitt-Ulms ◽

Rohan N. Krajeski ◽

Justin Lim ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

High Efficiency ◽

End Joining ◽

Differentiated Cells ◽

Dividing Cells ◽

Gene Integration ◽

Fluorescent Tagging ◽

Drag And Drop

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

10.1101/169441 ◽

2017 ◽

Cited By ~ 4

Author(s):

Ryan Marshall ◽

Colin S. Maxwell ◽

Scott P. Collins ◽

Michelle L. Luo ◽

Thomas Jacobsen ◽

...

Keyword(s):

Protein Purification ◽

Dna Cleavage ◽

Genome Engineering ◽

Gene Repression ◽

Live Cells ◽

Translation System ◽

Guide Rna ◽

E Coli ◽

Translation Systems

ABSTRACTCRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

Ecology Shapes Microbial Immune Strategy: Temperature and Oxygen as Determinants of the Incidence of CRISPR Adaptive Immunity

10.1101/326330 ◽

2018 ◽

Author(s):

Jake L. Weissman ◽

Rohan M. R. Laljani ◽

William F. Fagan ◽

Philip L. F. Johnson

Keyword(s):

Predictive Model ◽

End Joining ◽

Viral Pathogens ◽

Defense Strategies ◽

Machine Learning Approach ◽

Associated Proteins ◽

Using Data ◽

Restriction Modification

AbstractBacteria and archaea are locked in a near-constant battle with their viral pathogens. Despite previous mechanistic characterization of numerous prokaryotic defense strategies, the underlying ecological drivers of different strategies remain largely unknown and predicting which species will take which strategies remains a challenge. Here, we focus on the CRISPR immune strategy and develop a phylogenetically-corrected machine learning approach to build a predictive model of CRISPR incidence using data on over 100 traits across over 2600 species. We discover a strong but hitherto-unknown negative interaction between CRISPR and aerobicity, which we hypothesize may result from interference between CRISPR associated proteins and non-homologous end-joining DNA repair due to oxidative stress. Our predictive model also quantitatively confirms previous observations of an association between CRISPR and temperature. Finally, we contrast the environmental associations of different CRISPR system types (I, II, III) and restriction modification systems, all of which act as intracellular immune systems.

NHJ-1 regulates canonical non-homologous end joining in Caenorhabditis elegans

10.1101/763235 ◽

2019 ◽

Author(s):

Aleksandar Vujin ◽

Steven J. Jones ◽

Monique Zetka

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dapi Staining ◽

Strand Breaks ◽

C Elegans ◽

Meiotic Dsbs ◽

Ring Complex ◽

AbstractCanonical non-homologous end joining (cNHEJ) is a near-universally conserved pathway for the repair of DNA double-strand breaks (DSBs). While the cNHEJ pathway encompasses more than a dozen factors in vertebrates and is similarly complex in other eukaryotes, in the nematode C. elegans the entire known cNHEJ toolkit consists of two proteins that comprise the Ku ring complex, cku-70 and cku-80, and the terminal ligase lig-4. Here, we report the discovery of nhj-1 as the fourth cNHEJ factor in C. elegans. Observing a difference in the phenotypic response to ionizing radiation (IR) between two lines of the wild type N2 strain, we mapped the locus causative of IR-sensitivity to a candidate on chromosome V. Using CRISPR-Cas9 mutagenesis, we show that disrupting the nhj-1 sequence induces IR-sensitivity in an IR-resistant background. Double mutants of nhj-1 and the cNHEJ factors lig-4 or cku-80 do not exhibit additive IR-sensitivity, arguing that nhj-1 is a member of the cNHEJ pathway. Furthermore, like the loss of lig-4, the loss of nhj-1 in the com-1 genetic background, in which meiotic DSBs are repaired by cNHEJ instead of homologous recombination, increased the number of DAPI-staining bodies in diakinesis, consistent with increased chromosome fragmentation in the absence of cNHEJ repair. Finally, we show that NHJ-1 localizes to many somatic nuclei in the L1 larva, but not the primordial germline, which is in accord with a role in the predominantly somatically active cNHEJ. Although nhj-1 shares no sequence homology with other known eukaryotic cNHEJ factors and is taxonomically restricted to the Rhadbitid family, its discovery underscores the evolutionary plasticity of even highly conserved pathways, and may represent a springboard for further characterization of cNHEJ in C. elegans.

CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli

10.1101/605246 ◽

2019 ◽

Author(s):

Chaoyong Huang ◽

Tingting Ding ◽

Jingge Wang ◽

Xueqin Wang ◽

Jialei Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Genetic Engineering ◽

Double Strand Breaks ◽

Effective Strategy ◽

End Joining ◽

Strand Breaks ◽

E Coli ◽

Genomic Editing

AbstractUnlike eukaryotes, bacteria are less proficient in homologous recombination (HR) and non-homologous end joining (NHEJ). All existing genomic editing methods for Escherichia coli rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in chromosomal engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient knocking-out of E. coli genomic sequence of up to 83 kb. Moreover, the positive rate and editing efficiency is independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9 complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.SignificanceThe application in prokaryotes is difficult because of the weak homologous recombination and non-homologous end joining systems. E. coli, as the most-used prokaryote in metabolic engineering, has no NHEJ system. All existing genomic editing methods for E. coli rely on exogenous HR or NHEJ systems to repair double-strand breaks introduced by CRISPR/Cas9. In this report, we firstly demonstrate that the weak and previously ignored end-joining mechanism in E. coli can be used for efficient large-scale genetic engineering assisted by CRISPR/Cas9. Our efforts greatly simplify the genomic editing procedure of E. coli and provide an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

DNMT3b Dysfunction Promotes DNA cleavages at Centromeric R-loops to Increase Centromere Instability

10.1101/2020.06.04.133272 ◽

2020 ◽

Author(s):

Hsueh-Tzu Shih ◽

Wei-Yi Chen ◽

Hsin-Yen Wang ◽

Hsien-Da Huang ◽

Chih-Hung Chou ◽

...

Keyword(s):

Dna Methyltransferase ◽

Genome Instability ◽

Loss Of Function ◽

Dna Breaks ◽

End Joining ◽

Critical Function ◽

Satellite Sequences ◽

Mitotic Aberration ◽

Error Prone Repair ◽

ABSTRACTThis study investigates how DNA methyltransferase 3b (DNMT3b) dysfunction causes genome instability. We showed that in DNMT3b deficient cells, R-loops contribute to prominent γH2AX signal, which was mapped to repetitive satellite sequences including centromere regions. By ChIP and DRIP analyses, our data revealed that centromeric R-loops in DNMT3b deficient cells are removed by XPG/XPF, thus generating DNA breaks in centromeres to increase mitotic aberration. In immunodeficiency-centromeric instability-facial anomalies (ICF) patient cells carrying the loss-of-function mutation at DNMT3b, knockdown of XPG/XPF in ICF cells also reduces DNA breaks in centromere while bringing up centromeric R-loop to the level similar to that in wild-type cells. These results suggest that DNMT3b has a critical function in preventing XPG/XPF-mediated cleavages at centromeric R-loop sites. Finally, we showed the involvement of non-homologous end-joining repair at centromeric sites in ICF cells. Thus, DNA cleavages at centromeric R-loops with error-prone repair undermine centromere stability in ICF cells.

Variation among Metschnikowia pulcherrima Isolates for Genetic Modification and Homologous Recombination

Microorganisms ◽

10.3390/microorganisms9020290 ◽

2021 ◽

Vol 9 (2) ◽

pp. 290

Author(s):

Mauro Moreno-Beltrán ◽

Deborah Gore-Lloyd ◽

Christopher Chuck ◽

Daniel Henk

Keyword(s):

Homologous Recombination ◽

Genetic Modification ◽

Low Cost ◽

End Joining ◽

Recombination Rates ◽

Transformation Protocol ◽

Metschnikowia Pulcherrima ◽

Biotechnological Processes ◽

Different Strains

Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in biotechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homologous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 locus, suggesting that the genome sequence or structure alters the efficacy of this system.

CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera

International Journal of Molecular Sciences ◽

10.3390/ijms21103727 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3727

Author(s):

Sandugash Ibragimova ◽

Csilla Szebenyi ◽

Rita Sinka ◽

Elham I. Alzyoud ◽

Mónika Homa ◽

...

Keyword(s):

Genetic Manipulation ◽

Pathogenic Fungus ◽

Strand Break ◽

Targeted Mutagenesis ◽

End Joining ◽

Targeted Gene Disruption ◽

Lichtheimia Corymbifera ◽

Opportunistic Pathogenic

Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5′-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.