scholarly journals High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Haining Zhong ◽  
Cesar C Ceballos ◽  
Crystian I Massengill ◽  
Michael A Muniak ◽  
Lei Ma ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Gene Editing ◽  
Target Gene ◽  
Somatic Cells ◽  
High Fidelity ◽  
End Joining ◽  
Process Error ◽  
Endogenous Proteins ◽  
Non Homologous End Joining

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

scholarly journals High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

2020 ◽  
Author(s):  
Haining Zhong ◽  
Crystian I. Massengill ◽  
Michael A. Muniak ◽  
Lei Ma ◽  
Maozhen Qin ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Gene Editing ◽  
Target Gene ◽  
Somatic Cells ◽  
High Fidelity ◽  
End Joining ◽  
Process Error ◽  
Endogenous Proteins ◽  
Non Homologous End Joining

ABSTRACTPrecise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

scholarly journals Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids

2019 ◽  
Author(s):  
Justin A. Bosch ◽  
Ryan Colbeth ◽  
Jonathan Zirin ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Germ Line ◽  
Insertion Site ◽  
End Joining ◽  
Endogenous Proteins ◽  
Non Homologous End Joining ◽  
Universal Donor

AbstractTargeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. This method is a simple and fast alternative to HDR for certain strategies such as C-terminal tagging and gene disruption. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-RFP into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Finally, we apply this fast knock-in method to uncharacterized small open reading frame (smORF) genes. Therefore, homology-independent insertion is a useful genome editing technique in Drosophila that will better enable researchers to dissect gene function.Article summaryWe report a fast and simple genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. We demonstrate its usefulness in cultured cells to fluorescently tag endogenous proteins and in the fly germ line to generate heritable insertions that disrupt gene function and can act as expression reporters.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

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

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

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

2021 ◽  
Author(s):  
Tomas Strucko ◽  
Niklas L Andersen ◽  
Mikkel R Mahler ◽  
José L Martínez ◽  
Uffe H Mortensen
Keyword(s):  
Gene Editing ◽  
Point Mutations ◽  
Debaryomyces Hansenii ◽  
Cell Factory ◽  
Full Potential ◽  
Dna Breaks ◽  
End Joining ◽  
Dna Oligonucleotides ◽  
Non Homologous End Joining ◽  
Basic And Applied Research

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.

Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9

2021 ◽  
Author(s):  
Jun-Liang Tu ◽  
Xin-Yuan Bai ◽  
Yong-Liang Xu ◽  
Na Li ◽  
Jun-Wei Xu
Keyword(s):  
Homologous Recombination ◽  
Functional Genomics ◽  
Ganoderma Lucidum ◽  
Gene Disruption ◽  
Target Gene ◽  
End Joining ◽  
Gene Insertion ◽  
Replacement Method ◽  
Low Efficiency ◽  
Non Homologous End Joining

Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

scholarly journals COM1 , a factor of alternative non‐homologous end joining, lagging behind the classic non‐homologous end joining pathway in rice somatic cells

2020 ◽  
Vol 103 (1) ◽  
pp. 140-153
Author(s):  
Zhan Xu ◽  
Jianxiang Zhang ◽  
Xinjie Cheng ◽  
Yujie Tang ◽  
Zhiyun Gong ◽  
...  
Keyword(s):  
Somatic Cells ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jessica D. Gumerson ◽  
Amal Alsufyani ◽  
Wenhan Yu ◽  
Jingqi Lei ◽  
Xun Sun ◽  
...  
Keyword(s):  
Expression Vectors ◽  
End Joining ◽  
Reading Frame ◽  
Guide Rna ◽  
Domain Specific ◽  
Somatic Gene ◽  
Non Homologous End Joining ◽  
Cone Opsins ◽  
Inherited Retinal Degeneration

AbstractMutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

scholarly journals Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1091
Author(s):  
Aya Kurosawa
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Strand Break ◽  
In Vivo Studies ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

scholarly journals Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

2018 ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Non Homologous End Joining ◽  
Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

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