Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

/pdf/applications-of-genome-editing-technology-in-the-targeted-2giausc34s.pdf

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

https://hal.archives-ouvertes.fr/hal-02395971/document

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

The mammalian genome is constantly challenged by exogenous and endogenous threats. Although much is known about the mechanisms that maintain DNA and RNA integrity, we know surprisingly little about the mechanisms that underpin the pathology and tissue specificity of many disorders caused by defective responses to DNA or RNA damage. Of the different types of endogenous damage, protein-linked DNA breaks (PDBs) are emerging as an important player in cancer development and therapy. PDBs can arise during the abortive activity of DNA topoisomerases, a class of enzymes that modulate DNA topology during several chromosomal transactions, such as gene transcription and DNA replication, recombination and repair. In this Review, we discuss the mechanisms underpinning topoisomerase-induced PDB formation and repair with a focus on their role during gene transcription and the development of tissue-specific cancers.

Topoisomerase-mediated chromosomal break repair: an emerging player in many games

2596 Salivary gland tumors, a group of histologically diverse benign and malignant neoplasms, represent a challenging problem for diagnosis and treatment. A specific recurring chromosome translocation, t(11;19)(q21;p13), is associated with two types of salivary gland tumors, mucoepidermoid carcinomas and Warthin’s tumors. This translocation generates a fusion protein comprised of the N terminal CREB-binding domain of the CREB regulator MECT1 and the C terminal transcriptional activation domain of the Notch co-activator Mastermind-Like 2 (MAML2). Here we demonstrate that the MECT1-MAML2 fusion protein induces expression of multiple genes known to be CREB-mediated transcriptional targets. MECT1-MAML2 was found to bind to CREB, recruit p300/CBP into the CREB complex through a binding domain on MAML2, and constitutively activate CREB-dependent transcription. The transforming activity of MECT1-MAML2 was blocked by interfering with CREB DNA binding. Thus, this fusion oncogene functions to constitutively activate the cAMP signaling pathway. This study has identified a novel, critical mechanism of transformation for an oncogene associated very specifically with salivary gland tumors, and identified potential targets for the development of novel therapies.

Transforming activity of the MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation

Abstract Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them. 

/pdf/unintended-crispr-cas9-editing-outcomes-a-review-of-the-1jxo5c4j.pdf

Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells

Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

/pdf/zinc-finger-nuclease-induced-dna-double-stranded-breaks-and-3i6yps5uip.pdf

Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL.

The chromosome location, 11q21-23, is linked to loss of heterozygosity (LOH) in multiple tumors including those of breast, lung, and head and neck. To examine the process of LOH induction, the H292 cell line (human muco-epidermoid carcinoma) was irradiated or treated with anti-CD95 antibody, and individual clones isolated through two rounds of cloning. Regions of LOH were determined by screening a suite of eight polymorphic microsatellite markers covering 11p15-11q24 using fluorescent primers and genetic analyzer peak discrimination. LOH induction was observed extending through 11q21.1-11q23.3 in 6/49 of clones surviving 4 Gy and 8/50 after 8 Gy. Analysis of selected clones by Affymetrix 6.0 single nucleotide polymorphism (SNP) arrays confirmed the initial assessment indicating a consistent 27.3-27.7 Mbp deletion in multiple clones. The telomeric border of LOH mapped to a 1 Mbp region of elevated recombination. Whole genome analysis of SNP data indicated that site-restricted LOH also occurred across multiple additional genomic locations. These data indicate that 11q21.1-11q23.3, and potentially other regions of this cell line are sites of intrinsic cell-specific instability leading to LOH after irradiation. Such deletions may subsequently be propagated by genetic selection and clonal expansion.

11q21.1‐11q23.3 Is a site of intrinsic genomic instability triggered by irradiation

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

To Uyen Do

Author Tools

Chat about Author

Papers

Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL.

11q21.1‐11q23.3 Is a site of intrinsic genomic instability triggered by irradiation