scholarly journals A Crisper Look at Genome Editing: RNA-guided Genome Modification

2013 ◽  
Vol 21 (4) ◽  
pp. 720-722 ◽  
Author(s):  
Mara Damian ◽  
Matthew H Porteus
Keyword(s):  
Genome Editing ◽  
Genome Modification

scholarly journals CRISPR-Cas9: Role in Processing of Modular Metabolic Engineered Bio-Based Products

2021 ◽  
Author(s):  
Vishnu Sharma ◽  
Tarun Kumar Kumawat ◽  
Anjali Pandit ◽  
Bhoomika Sharma ◽  
Pooja Agarwal ◽  
...  
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Metabolic Product ◽  
Modifying Genes ◽  
Genome Modification ◽  
Foreign Dna ◽  
Genome Level

Biogenetic engineering is a significant technology to sensibly manage microbial metabolic product factories. Genome modification methods for efficiently controlling and modifying genes at the genome level have progressed in biogenetic engineering during the last decade. CRISPR is genome editing technology that allows for the modification of organisms’ genomes. CRISPR and its related RNA-guided endonuclease are versatile advanced immune system frameworks for defending against foreign DNA and RNAs. CRISPR is efficient, accessible, and trustworthy genomic modification tool in unparalleled resolution. At present, CRISPR-Cas9 method is expanded to industrially manipulate cells. Metabolically modified organisms are quickly becoming interested in the production of different bio-based components. Here, chapter explore about the control productivity of targeted biomolecules in divergent cells based on the use of different CRISPR-related Cas9.

scholarly journals Engineering and optimising deaminase fusions for genome editing

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Luhan Yang ◽  
Adrian W. Briggs ◽  
Wei Leong Chew ◽  
Prashant Mali ◽  
Marc Guell ◽  
...  
Keyword(s):  
Genome Editing ◽  
Binding Sites ◽  
Chromosomal Abnormalities ◽  
Whole Genome ◽  
Bacterial Cells ◽  
Site Specific ◽  
Cytidine Deaminases ◽  
Genome Modification ◽  
Dna Binding Affinity ◽  
Cytidine Deamination

Abstract Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

scholarly journals Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nora Grahl ◽  
Elora G. Demers ◽  
Alex W. Crocker ◽  
Deborah A. Hogan
Keyword(s):  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Promoter Activity ◽  
Protein Complexes ◽  
Content Type ◽  
Guide Rna ◽  
Genome Modification ◽  
Cas9 Protein ◽  
Rna Protein Complexes

ABSTRACT Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.

scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

scholarly journals Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

2021 ◽  
Author(s):  
Xiya Zhang ◽  
Tao Li ◽  
Jianping Ou ◽  
Junjiu Huang ◽  
Puping Liang
Keyword(s):  
Genome Editing ◽  
Disease Model ◽  
Laboratory Research ◽  
Animal Disease ◽  
End Joining ◽  
Mammalian Embryo ◽  
Genome Modification ◽  
Recent Developments ◽  
Mammalian Embryos ◽  
Non Homologous End Joining

AbstractRecent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.

scholarly journals Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pengpeng Liu ◽  
Shun-Qing Liang ◽  
Chunwei Zheng ◽  
Esther Mintzer ◽  
Yan G. Zhao ◽  
...  
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Tumor Formation ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Modification System ◽  
Adult Mice ◽  
Genome Modification ◽  
Split Intein

AbstractPrime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

scholarly journals From Gene Targeting to Genome Editing: Transgenic animals applications and beyond

2015 ◽  
Vol 87 (2 suppl) ◽  
pp. 1323-1348 ◽  
Author(s):  
MAURÍCIO ROCHA-MARTINS ◽  
GABRIEL R. CAVALHEIRO ◽  
GABRIEL E. MATOS-RODRIGUES ◽  
RODRIGO A.P. MARTINS
Keyword(s):  
Genome Editing ◽  
Biomedical Research ◽  
Transgenic Animals ◽  
Paradigm Shifts ◽  
New Techniques ◽  
Future Directions ◽  
Cellular Physiology ◽  
The Past ◽  
Genome Modification ◽  
Help Model

ABSTRACTGenome modification technologies are powerful tools for molecular biology and related areas. Advances in animal transgenesis and genome editing technologies during the past three decades allowed systematic interrogation of gene function that can help model how the genome influences cellular physiology. Genetic engineering via homologous recombination (HR) has been the standard method to modify genomic sequences. Nevertheless, nuclease-guided genome editing methods that were developed recently, such as ZFN, TALEN and CRISPR/Cas, opened new perspectives for biomedical research. Here, we present a brief historical perspective of genome modification methods, focusing on transgenic mice models. Moreover, we describe how new techniques were discovered and improved, present the paradigm shifts and discuss their limitations and applications for biomedical research as well as possible future directions.

scholarly journals Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

2020 ◽  
Author(s):  
Pengpeng Liu ◽  
Shun-Qing Liang ◽  
Chunwei Zheng ◽  
Esther Mintzer ◽  
Yan G. Zhao ◽  
...  
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Tumor Formation ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Modification System ◽  
Adult Mice ◽  
Genome Modification ◽  
Split Intein

AbstractPrime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

scholarly journals Germline genome modification through novel political, ethical, and social lenses

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009741
Author(s):  
Vicki Xafis ◽  
G. Owen Schaefer ◽  
Markus K. Labude ◽  
Yujia Zhu ◽  
Soren Holm ◽  
...  
Keyword(s):  
Clinical Research ◽  
Genome Editing ◽  
Scientific Community ◽  
National Interest ◽  
Research Application ◽  
Structural Injustice ◽  
Genome Modification ◽  
Strong Focus ◽  
Societal Consensus

Much has been written about gene modifying technologies (GMTs), with a particularly strong focus on human germline genome editing (HGGE) sparked by its unprecedented clinical research application in 2018, shocking the scientific community. This paper applies political, ethical, and social lenses to aspects of HGGE to uncover previously underexplored considerations that are important to reflect on in global discussions. By exploring 4 areas—(1) just distribution of HGGE benefits through a realist lens; (2) HGGE through a national interest lens; (3) “broad societal consensus” through a structural injustice lens; and (4) HGGE through a scientific trustworthiness lens—a broader perspective is offered, which ultimately aims to enrich further debates and inform well-considered solutions for developments in this field. The application of these lenses also brings to light the fact that all discussions about scientific developments involve a conscious or unconscious application of a lens that shapes the direction of our thinking.

scholarly journals Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wen Xu ◽  
Wei Song ◽  
Yongxing Yang ◽  
Ying Wu ◽  
Xinxin Lv ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
High Fidelity ◽  
Knock Out ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Modification ◽  
Targeted Genome Modification ◽  
Target Effect

Abstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. Conclusions We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

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