A CRISPR/Cas9 Method Facilitates Efficient Oligo-Mediated Gene Editing in Debaryomyces Hansenii

Abstract The halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques does not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism pose further challenges when precise gene targeting is required. In this study, we present a novel plasmid based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple sgRNAs that provides possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed an NHEJ deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single stranded DNA oligonucleotides are sufficient to direct repair of DNA breaks induced by sgRNA-Cas9 resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

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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 ◽

Non Homologous End Joining

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.

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Editing livestock genomes with site-specific nucleases

Reproduction Fertility and Development ◽

10.1071/rd13260 ◽

2014 ◽

Vol 26 (1) ◽

pp. 74 ◽

Cited By ~ 13

Author(s):

Daniel F. Carlson ◽

Wenfang Tan ◽

Perry B. Hackett ◽

Scott C. Fahrenkrug

Keyword(s):

Genetic Alterations ◽

Large Animal ◽

Dna Breaks ◽

Transcription Activator ◽

End Joining ◽

Site Specific ◽

Homology Directed Repair ◽

Double Strand Dna Breaks ◽

Non Homologous End Joining ◽

Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

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Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

Nature Communications ◽

10.1038/s41467-021-27004-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Aron Ferenczi ◽

Yen Peng Chew ◽

Erika Kroll ◽

Charlotte von Koppenfels ◽

Andrew Hudson ◽

...

Keyword(s):

Dna Repair ◽

Chlamydomonas Reinhardtii ◽

Nuclear Dna ◽

Model Organism ◽

Single Strand ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Non Homologous End Joining ◽

Underlying Mechanisms

AbstractSingle-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

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Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

Plant Biotechnology ◽

10.5511/plantbiotechnology.21.0407a ◽

2021 ◽

Author(s):

Marcos Fernando Basso ◽

Karoline Estefani Duarte ◽

Thais Ribeiro Santiago ◽

Wagner Rodrigo de Souza ◽

Bruno de Oliveira Garcia ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Gene Knockout ◽

Setaria Viridis ◽

End Joining ◽

Non Homologous End Joining

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DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

10.1101/603381 ◽

2019 ◽

Author(s):

Alexander Smirnov ◽

Anastasia Yunusova ◽

Alexey Korablev ◽

Irina Serova ◽

Veniamin Fishman ◽

...

Keyword(s):

Homologous Recombination ◽

Dna Molecules ◽

Dna Breaks ◽

End Joining ◽

Exogenous Dna ◽

Transgenic Organisms ◽

Mouse Zygote ◽

Non Homologous End Joining ◽

Transgenic Embryos ◽

Double Holliday Junction

AbstractMechanisms that ensure repair of double-stranded DNA breaks play a key role in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found in the form of concatemer consisting of multiple co-integrated transgene copies. Here we investigated contribution of various DSB repair pathways to the concatemer formation. We injected a pool of linear DNA molecules carrying unique barcodes at both ends into mouse zygotes and obtained 10 transgenic embryos with transgene copy number ranging from 1 to 300 copies. Sequencing of the barcodes allowed us to assign relative positions to the copies in concatemers and to detect recombination events that happened during integration. Cumulative analysis of approximately 1000 integrated copies revealed that more than 80% of copies underwent recombination when their linear ends were processed by SDSA or DSBR. We also observed evidence of double Holliday junction (dHJ) formation and crossing-over during the formation of concatemers. Additionally, sequencing of indels between copies showed that at least 10% of the DNA molecules introduced into the zygote are ligated by non-homologous end joining (NHEJ). Our barcoding approach documents high activity of homologous recombination after exogenous DNA injection in mouse zygote.

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ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining

Oncogene ◽

10.1038/s41388-019-1028-7 ◽

2019 ◽

Vol 39 (4) ◽

pp. 754-766 ◽

Cited By ~ 7

Author(s):

Sara Nicolai ◽

Robert Mahen ◽

Giuseppe Raschellà ◽

Alberto Marini ◽

Marco Pieraccioli ◽

...

Keyword(s):

Dna Damage ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Dna Lesions ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Nhej Repair ◽

Non Homologous End Joining

Abstract Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients’ stratification and for the development of personalised therapeutic strategies.

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Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components

Gene Therapy ◽

10.1038/s41434-019-0119-y ◽

2020 ◽

Vol 27 (5) ◽

pp. 209-225 ◽

Cited By ~ 6

Author(s):

Ignazio Maggio ◽

Hidde A. Zittersteijn ◽

Qian Wang ◽

Jin Liu ◽

Josephine M. Janssen ◽

...

Keyword(s):

Dna Cleavage ◽

Adenoviral Vector ◽

Gene Editing ◽

Nuclear Translocation ◽

Viral Vector ◽

High Specificity ◽

Dna Breaks ◽

End Joining ◽

Nuclear Localization Signals ◽

And Performance

AbstractEnhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

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Precision genome editing in the CRISPR era

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0137 ◽

2017 ◽

Vol 95 (2) ◽

pp. 187-201 ◽

Cited By ~ 49

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.

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MMEJ-based Precision Gene Editing for applications in Gene Therapy and Functional Genomics

10.1101/2020.04.25.060541 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gabriel Martínez-Gálvez ◽

Armando Manduca ◽

Stephen C. Ekker

Keyword(s):

Gene Editing ◽

Reverse Genetics ◽

Embryonic Stem ◽

Strand Break ◽

Loss Of Function ◽

End Joining ◽

Dsb Repair ◽

Therapeutic Applications ◽

Predictive Algorithm ◽

Non Homologous End Joining

ABSTRACTExperiments in gene editing commonly elicit error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. MENTHU is a computational tool that predicts nuclease-targetable sites likely to result in MMEJ-repaired, homogeneous genotypes (PreMAs) in zebrafish. We deployed MENTHU on 5,885 distinct Cas9-mediated DSBs in mouse embryonic stem cells, and compared the predictions to those by inDelphi, another DSB repair predictive algorithm. MENTHU correctly identified 46% of all PreMAs available, doubling the sensitivity of inDelphi. We also introduce MENTHU@4, an MENTHU update trained on this large dataset. We trained two MENTHU-based algorithms on this larger dataset and validated them against each other, MENTHU, and inDelphi. Finally, we estimated the frequency and distribution of SpCas9-targetable PreMAs in vertebrate coding regions to evaluate MMEJ-based targeting for gene discovery. 44 out of 54 genes (81%) contained at least one early out-of-frame PreMA and 48 out of 54 (89%) did so when also considering Cas12a. We suggest that MMEJ can be deployed at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

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CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

10.1101/2020.10.05.327569 ◽

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.

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