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

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.

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Template-independent genome editing and repairing correct frameshift disease in vivo

10.1101/2020.11.13.381160 ◽

2020 ◽

Author(s):

Lian Liu ◽

Kuan Li ◽

Linzhi Zou ◽

Hanqing Hou ◽

Qun Hu ◽

...

Keyword(s):

Therapeutic Strategy ◽

Frameshift Mutation ◽

Therapeutic Potential ◽

Scale Up ◽

End Joining ◽

Reading Frame ◽

Mendelian Disorders ◽

Non Homologous End Joining ◽

Therapy Strategies

AbstractFrameshift mutation caused by small insertions/deletions (indels) often generate truncated and non-functional proteins, which underlies 22% inherited Mendelian disorders in humans. However, there is no efficient in vivo gene therapy strategies available to date, especially in postmitotic systems. Here, we leveraged the non-homologous end joining (NHEJ) mediated non-random editing profiles to compensate the frameshift mutation in a USH1F mouse model – av3j. After treatment by the selected gRNA, about 50% editing products showed reading-frame restoration, and more than 70% targeted hair cells recovered mechanotransduction. In vivo treatment ameliorated the hearing and balance symptoms in homozygous mutant mice. Furthermore, a scale-up analysis of 114 gRNAs targeting 40 frameshift deafness mutations reveals that 65% loci have at least one gRNA with predicted therapeutic potential. Together, our study demonstrates that the NHEJ-mediated frame restoration is a simple and highly efficient therapeutic strategy for small-indel induced frameshift mutations.

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

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

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.

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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

10.1101/358424 ◽

2018 ◽

Cited By ~ 2

Author(s):

Wannaporn Ittiprasert ◽

Victoria H. Mann ◽

Shannon E. Karinshak ◽

Avril Coghlan ◽

Gabriel Rinaldi ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Genome Editing ◽

Human Macrophage ◽

Wild Type ◽

End Joining ◽

Chromosomal Breakage ◽

Knock Out ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

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Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2017.0799 ◽

2017 ◽

Vol 284 (1860) ◽

pp. 20170799 ◽

Cited By ~ 58

Author(s):

Thomas A. A. Prowse ◽

Phillip Cassey ◽

Joshua V. Ross ◽

Chandran Pfitzner ◽

Talia A. Wittmann ◽

...

Keyword(s):

Female Sterility ◽

Gene Drive ◽

End Joining ◽

Guide Rna ◽

Deleterious Alleles ◽

Precise Knowledge ◽

Animal Populations ◽

Individual Based Models ◽

Non Homologous End Joining ◽

Gene Drives

Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial ‘silver bullet’ for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their efficacy for controlling wild populations is threatened by inherent polymorphic resistance and the creation of resistance alleles via non-homologous end-joining (NHEJ)-mediated DNA repair. We used stochastic individual-based models to identify realistic gene-drive strategies capable of eradicating vertebrate pest populations (mice, rats and rabbits) on islands. One popular strategy, a sex-reversing drive that converts heterozygous females into sterile males, failed to spread and required the ongoing deployment of gene-drive carriers to achieve eradication. Under alternative strategies, multiplexed gRNAs could overcome inherent polymorphic resistance and were required for eradication success even when the probability of NHEJ was low. Strategies causing homozygotic embryonic non-viability or homozygotic female sterility produced high probabilities of eradication and were robust to NHEJ-mediated deletion of the DNA sequence between multiplexed endonuclease recognition sites. The latter two strategies also purged the gene drive when eradication failed, therefore posing lower long-term risk should animals escape beyond target islands. Multiplexing gRNAs will be necessary if this technology is to be useful for insular extirpation attempts; however, precise knowledge of homing rates will be required to design low-risk gene drives with high probabilities of eradication success.

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Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase

Genes ◽

10.3390/genes12071091 ◽

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.

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CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion

Nature Communications ◽

10.1038/s41467-021-22927-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhiqian Li ◽

Nimi Marcel ◽

Sushil Devkota ◽

Ankush Auradkar ◽

Stephen M. Hedrick ◽

...

Keyword(s):

Gene Conversion ◽

Somatic Mutations ◽

Genetic Screens ◽

End Joining ◽

Genetic Elements ◽

Homology Directed Repair ◽

Somatic Gene ◽

Non Homologous End Joining ◽

Or Gene ◽

Therapy Strategies

AbstractCRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.

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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

10.1101/300723 ◽

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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Leaky severe combined immunodeficiency in mice lacking non-homologous end joining factors XLF and MRI

10.1101/2020.03.04.976829 ◽

2020 ◽

Author(s):

Sergio Castañeda-Zegarra ◽

Qindong Zhang ◽

Amin Alirezaylavasani ◽

Marion Fernandez-Berrocal ◽

Rouan Yao ◽

...

Keyword(s):

Severe Combined Immunodeficiency ◽

Embryonic Lethality ◽

Combined Immunodeficiency ◽

End Joining ◽

Mild Phenotype ◽

Reduced Body ◽

Non Homologous End Joining ◽

Mature Lymphocyte ◽

And Function

AbstractNon-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

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Linear-double-stranded DNA (ldsDNA) based AND logic computation in mammalian cells

10.1101/266056 ◽

2018 ◽

Author(s):

Weijun Su ◽

Chunze Zhang ◽

Shuai Li

Keyword(s):

Synthetic Biology ◽

Mammalian Cells ◽

Rapid Development ◽

Genetic Circuits ◽

End Joining ◽

Double Stranded Dna ◽

Logic Computation ◽

Key Enzyme ◽

Non Homologous End Joining

AbstractSynthetic biology employs engineering principles to redesign biological system for clinical or industrial purposes. The development and application of novel genetic devices for genetic circuits construction will facilitate the rapid development of synthetic biology. Here we demonstrate that mammalian cells could perform two- and three-input linear-double-stranded DNA (ldsDNA) based Boolean AND logic computation. Through hydrodynamic ldsDNA delivery, two-input ldsDNA-base AND-gate computation could be achieved in vivo. Inhibition of DNA-PKcs expression, a key enzyme in non-homologous end joining (NHEJ), could significantly downregulate the intensity of output signals from ldsDNA-based AND-gate. We further reveal that in mammalian cells ldsDNAs could undergo end processing and then perform AND-gate calculation to generate in-frame output proteins. Moreover, we show that ldsDNAs or plasmids with identical overlapping sequences could also serve as inputs of AND-gate computation. Our work establishes novel genetic devices and principles for genetic circuits construction, thus may open a new gate for the development of new disease targeting strategies and new protein genesis methodologies.

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