NUDT16L1 (TIRR) knockdown increases genome editing efficacy

2021 ◽  
pp. 3-10
Author(s):  
А.А. Анучина ◽  
С.А. Смирнихина ◽  
М.И. Зайнитдинова ◽  
А.Г. Демченко ◽  
О.С. Мишина ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Small Interfering Rnas ◽  
Repair Pathway ◽  
Major Repair ◽  
Homology Directed Repair ◽  
Pathogenic Mutations ◽  
First Time ◽  
Egfp Signal

Введение. Эффективная точная коррекция мутаций очень важна для внедрения CRISPR-Cas в качестве инструмента для потенциальной генной терапии. Механизм направленной гомологичной репарации (НГР), активируемый после внесения нуклеазой двунитевого разрыва, позволяет корректировать все существующие типы мутаций. Однако НГР не является доминирующим механизмом в клетке, что ограничивает ее эффективность. В нашем исследовании впервые описано, как фактор репарации NUDT16L1 может контролировать эффективность НГР при редактировании с помощью CRISPR-Cas. Цель: исследовать влияние нокдауна и гиперэкспрессии NUDT16L1 на эффективность НГР. Методы. Исследование проводили на клеточной культуре HEK293T. Систему CRISPR-Cas в плазмиде, а также вектор для гиперэкспрессии NUDT16L1 доставляли путём липофекции. Для нокдауна гена NUDT16L1 использовали малые интерферирующие РНК. Результаты. Нокдаун NUDT16L1 повышает уровень НГР в плазмидном и геномном локусах, что проявляется повышением доли GFP-позитивных клеток в 1,8-3,6 раз. Заключение. Нокдаун гена NUDT16L1 может быть использован для повышения эффективности исправления патогенных вариантов ДНК методом геномного редактирования. Introduction. Effective precise knock-in is crucial for implementing CRISPR-Cas9 system as an efficient instrument for potential gene therapy. Homology directed repair (HDR) pathway allows correction of all types of existing mutations. However, HDR is not a major repair pathway of the cell that limits its efficiency. In our study, we present for the first time how repair factors NUDT16L1 controls HDR efficiency. Aim: to study an influence of NUDT16L1 knockdown and overexpression on the HDR efficacy. Methods. HEK293T culture was used to perform the research. Plasmid CRISPR-Cas system along with NUDT16L1 overexpression vector were delivered with lipofection. For NUDT16L1 knockdown small interfering RNAs were used. Results. We discovered that knockdown of NUDT16L1 enhances HDR both in the plasmid and genomic loci increasing eGFP signal from 1.8 to 3.6 times in HEK293T cells. Conclusion. NUDT16L1 knockdown could be used for enhancing of the pathogenic mutations correction through genome editing.

scholarly journals 568. Transient Manipulation of DNA Damage Repair Pathway Choice Improves Homology-Directed Repair During CRISPR/Cas9-Mediated Genome Editing

2016 ◽  
Vol 24 ◽  
pp. S227
Author(s):  
Pankaj K. Mandal ◽  
Bruna S. Paulsen ◽  
Richard Frock ◽  
Paula Gutierrez-Martinez ◽  
Wataru Ebina ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Editing ◽  
Dna Damage Repair ◽  
Repair Pathway ◽  
Homology Directed Repair ◽  
Damage Repair ◽  
Pathway Choice

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 Progress in Gene Therapy to Prevent Retinal Ganglion Cell Loss in Glaucoma and Leber’s Hereditary Optic Neuropathy

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Sara E. Ratican ◽  
Andrew Osborne ◽  
Keith R. Martin
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Optic Nerve ◽  
Genome Editing ◽  
Optic Neuropathy ◽  
Single Gene ◽  
Leber’S Hereditary Optic Neuropathy ◽  
Leber's Hereditary Optic Neuropathy ◽  
Potential Use ◽  
Hereditary Optic Neuropathy

The eye is at the forefront of the application of gene therapy techniques to medicine. In the United States, a gene therapy treatment for Leber’s congenital amaurosis, a rare inherited retinal disease, recently became the first gene therapy to be approved by the FDA for the treatment of disease caused by mutations in a specific gene. Phase III clinical trials of gene therapy for other single-gene defect diseases of the retina and optic nerve are also currently underway. However, for optic nerve diseases not caused by single-gene defects, gene therapy strategies are likely to focus on slowing or preventing neuronal death through the expression of neuroprotective agents. In addition to these strategies, there has also been recent interest in the potential use of precise genome editing techniques to treat ocular disease. This review focuses on recent developments in gene therapy techniques for the treatment of glaucoma and Leber’s hereditary optic neuropathy (LHON). We discuss recent successes in clinical trials for the treatment of LHON using gene supplementation therapy, promising neuroprotective strategies that have been employed in animal models of glaucoma and the potential use of genome editing techniques in treating optic nerve disease.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

scholarly journals Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz0051 ◽  
Author(s):  
Xinyu Ling ◽  
Bingteng Xie ◽  
Xiaoqin Gao ◽  
Liying Chang ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Specific Chemical ◽  
Site Specific ◽  
Additional Tool ◽  
Homology Directed Repair ◽  
Chemically Modified ◽  
Dna Template ◽  
Oligonucleotide Conjugates

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

scholarly journals Genome Editing Strategies to Treat Beta-Hemoglobinopathies

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-16-SCI-16
Author(s):  
Mitchell J Weiss
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Great Promise ◽  
Globin Genes ◽  
Hematopoietic Stem ◽  
Medical Therapies

Genetic forms of anemia caused by HBB gene mutations that impair beta globin production are extremely common worldwide. The resultant disorders, mainly sickle cell disease (SCD) and beta-thalassemia, cause substantial morbidity and early mortality. Treatments for these diseases include medical therapies and bone marrow transplantation (BMT), which can be curative. However, medical therapies are suboptimal and BMT is associated with serious toxicities, particularly because HLA-matched allogeneic sibling donors are not available for most patients. Thus, new therapies are urgently needed for millions of affected individuals. Gene therapy offers great promise to cure SCD and beta thalassemia and emerging genome editing technologies represent a new form of gene therapy. Approaches to cure SCD and beta-thalassemia via genome editing include: 1) Correction of HBB mutations by homology directed repair (HDR); 2) use of non-homologous end joining (NHEJ) to activate gamma globin production and raise fetal hemoglobin (HbF) levels; 3) NHEJ to disrupt alpha-globin genes (HBA1 or HBA2) and thereby alleviate globin chain imbalance in intermediately severe forms of beta thalassemia. Challenges for these approaches include selection of the most effective genome editing tools, optimizing their delivery to hematopoietic stem cells (HSCs), improving specificity and better understanding potential off target effects, particularly those that are biologically relevant. Technologies for genome editing are advancing rapidly and being tested in preclinical models for HBB-mutated disorders. Ultimately, however, the best strategies can only be identified in clinical trials. This will require close collaborations between basic/translational researchers who study genome editing, clinical hematologists and collaboration between experts in academia and the bio-pharmaceutical industry. Disclosures No relevant conflicts of interest to declare.

Recent progress in genome editing for gene therapy applications: the French perspective

2021 ◽  
Author(s):  
Mario Amendola ◽  
Aurélie Bedel ◽  
Ana Buj Bello ◽  
Mathieu Carrara ◽  
Jean-paul Concordet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Recent Progress

scholarly journals Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1318 ◽  
Author(s):  
Nadja Bischoff ◽  
Sandra Wimberger ◽  
Marcello Maresca ◽  
Cord Brakebusch
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Animal Models ◽  
Molecular Biology ◽  
Small Molecules ◽  
Genome Editing ◽  
Drug Targets ◽  
Targeted Mutagenesis ◽  
Dna Repair Mechanisms ◽  
Repair Mechanisms

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

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