Homing endonucleases: DNA scissors on a mission

Genome ◽  
2012 ◽  
Vol 55 (8) ◽  
pp. 553-569 ◽  
Author(s):  
Mohamed Hafez ◽  
Georg Hausner
Keyword(s):  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Transcription Activator ◽  
Group I ◽  
Gene Knockouts ◽  
Or Gene ◽  
Triplex Forming Oligonucleotide ◽  
Self Splicing ◽  
Specific Sequences

Buried within the genomes of many microorganisms are genetic elements that encode rare-cutting homing endonucleases that assist in the mobility of the elements that encode them, such as the self-splicing group I and II introns and in some cases inteins. There are several different families of homing endonucleases and their ability to initiate and target specific sequences for lateral transfers makes them attractive reagents for gene targeting. Homing endonucleases have been applied in promoting DNA modification or genome editing such as gene repair or “gene knockouts”. This review examines the categories of homing endonucleases that have been described so far and their possible applications to biotechnology. Strategies to engineer homing endonucleases to alter target site specificities will also be addressed. Alternatives to homing endonucleases such as zinc finger nucleases, transcription activator-like effector nucleases, triplex forming oligonucleotide nucleases, and targetrons are also briefly discussed.

Genome Editing Toolbox

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-11-SCI-11
Author(s):  
Andrew M. Scharenberg
Keyword(s):  
Genome Engineering ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Practical Implementation ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Dna Breaks ◽  
Transcription Activator ◽  
Advisory Committees ◽  
Core Technology

Abstract Nucleases capable of making targeted breaks in genomic DNA are a core technology required for genome engineering, an emerging field of technology for making precise alterations in cellular genomes. Over the past ten years, four major platforms have emerged for generation of nucleases able to make targeted DNA breaks with a high degree of efficiency and specificity: homing endonucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and RNA-guided nucleases. This talk will cover the biochemistry and platform-specific attributes of each type of nuclease, along with evolution/improvements in nucleases and related technologies and aspects of the practical implementation of nuclease technology for gene knockout and gene repair in primary hematopoietic cells. Disclosures Scharenberg: Pregenen Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cellectis therapeutics: Consultancy.

scholarly journals A Self-Splicing Group I Intron in DNA Polymerase Genes of T7-Like Bacteriophages

2004 ◽  
Vol 186 (23) ◽  
pp. 8153-8155 ◽  
Author(s):  
Richard P. Bonocora ◽  
David A. Shub
Keyword(s):  
Dna Polymerase ◽  
Group I Intron ◽  
Group I Introns ◽  
Homing Endonucleases ◽  
Structural Element ◽  
Group I ◽  
Close Relatives ◽  
Self Splicing

ABSTRACT Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of ΦI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.

scholarly journals Update of Regulatory Options of New Breeding Techniques and Biosafety Approaches among Selected Countries: A Review

2020 ◽  
pp. 18-35
Author(s):  
Silas Obukosia ◽  
Olalekan Akinbo ◽  
Woldeyesus Sinebo ◽  
Moussa Savadogo ◽  
Samuel Timpo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetically Modified Organisms ◽  
Zinc Finger Nucleases ◽  
Intermediate Step ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Associated Proteins ◽  
Genomic Editing ◽  
New Breeding Techniques ◽  
Breeding Techniques

A new set of breeding techniques, referred to as New Breeding Techniques developed in the last two decades have potential for enhancing improved productivity in crop and animal breeding globally. These include site directed nucleases based genomic editing procedures-CRISPR and Cas associated proteins, Zinc Finger Nucleases, Meganucleases/Homing Endonucleases and Transcription- Activator Like-Effector Nucleases for genome editing and other technologies including- Oligonucleotide-Directed Mutagenesis, Cisgenesis and intragenesis, RNA-Dependent DNA methylation; Transgrafting, Agroinfiltration, Reverse breeding. There are ongoing global debates on whether the processes of and products emerging from these technologies should be regulated as genetically modified organisms or approved as conventional products. Decisions on whether to regulate as GMOs are based both on understanding of the molecular basis of their development and if the GMO intermediate step was used. For example- cisgenesis, can be developed using Agrobacterium tumefaciens methods of transformation, a process used by GMO but if the selection is properly conducted the intermediate GMO elements will be eliminated and the final product will be identical to the conventionally developed crops. Others like Site Directed Nuclease 3 are regulated as GMOs in countries such as United State of America, Canada, European Union, Argentina, Australia. Progress in genome editing research, testing of genome edited bacterial blight resistant rice, development of Guidelines for regulating new breeding techniques or genome editing in Africa is also covered with special reference to South Africa, Kenya and Nigeria. Science- and evidence-based approach to regulation of new breeding techniques among regulators and policy makers should be strongly supported.

scholarly journals CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

2021 ◽  
Vol 14 (11) ◽  
pp. 1171
Author(s):  
Sahar Serajian ◽  
Ehsan Ahmadpour ◽  
Sonia M. Rodrigues Oliveira ◽  
Maria de Lourdes Pereira ◽  
Siamak Heidarzadeh
Keyword(s):  
Infectious Diseases ◽  
Clinical Microbiology ◽  
Gene Editing ◽  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Practical Applications ◽  
Novel Technologies ◽  
Resistant Strains ◽  
Drug Resistant Strains

Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.

Genome modifications in crops employing engineered nucleases

2016 ◽  
Author(s):  
Harshvardhan N. Zala ◽  
Tejas C. Bosamia ◽  
Yogesh M. Shukla ◽  
Sushil Kumar ◽  
Kalyani S. Kulkarni
Keyword(s):  
Genetically Modified Organisms ◽  
Crop Improvement ◽  
Zinc Finger Nucleases ◽  
World Population ◽  
Homing Endonucleases ◽  
Induced Mutations ◽  
Transcription Activator ◽  
Random Mutation ◽  
Engineered Nucleases ◽  
Genome Modifications

Crop improvement aims at substantial enhancements in the quality, yield and stress resistance of crops to meet the increasing food demand of growing world population. Targeted genome modification of crop plants is one of the ways to achieve this. This technology supersedes conventional methods limited by the inefficiencies of random mutation, accuracy and stability. It employs site-directed nucleases to create breaks at specific points in the target genome for desired alteration with high-precision. There are four nucleases namely, LAGLIDADG homing endonucleases (LHEs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) nucleases out of which three, ZFNs, TALENs and CRISPR have been highly studied and evaluated in various crop systems for economic trait. Potency of engineered nucleases lies in their efficacy to bring desired modification in diploid as well as in polyploid plant genomes. Modifications using genome editing are similar to natural or conventional method like induced mutations and are foreseen to waive regulatory actions as applicable to genetically modified organisms. This review seeks to emphasize on the employment of engineered nucleases in various crops plants till date.

scholarly journals CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications

2020 ◽  
Vol 21 (9) ◽  
pp. 3038 ◽  
Author(s):  
Xingbo Xu ◽  
Melanie S. Hulshoff ◽  
Xiaoying Tan ◽  
Michael Zeisberg ◽  
Elisabeth M. Zeisberg
Keyword(s):  
Transcriptional Activation ◽  
Gene Activation ◽  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Base Editing ◽  
Advantages And Disadvantages ◽  
Associated Proteins

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Organellar Introns in Fungi, Algae, and Plants

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2001
Author(s):  
Jigeesha Mukhopadhyay ◽  
Georg Hausner
Keyword(s):  
Gene Expression ◽  
Ribosomal Proteins ◽  
Heterologous Gene Expression ◽  
Group I Introns ◽  
Group Ii Introns ◽  
Homing Endonucleases ◽  
Organellar Genomes ◽  
Chloroplast Genomes ◽  
Group I ◽  
Group Ii

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

scholarly journals Mini-Review Regarding the Applicability of Genome Editing Techniques Developed for Studying Infertility

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 246
Author(s):  
Bogdan Doroftei ◽  
Ovidiu-Dumitru Ilie ◽  
Maria Puiu ◽  
Alin Ciobica ◽  
Ciprian Ilea
Keyword(s):  
Motor Activity ◽  
Experimental Model ◽  
Zinc Finger ◽  
Biomedical Research ◽  
Zinc Finger Nucleases ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Phenotypic Changes ◽  
Wide Range ◽  
Short Palindromic Repeat

Infertility is a highly debated topic today. It has been long hypothesized that infertility has an idiopathic cause, but recent studies demonstrated the existence of a genetic substrate. Fortunately, the methods of editing the human genome proven to be revolutionary. Following research conducted, we identified a total of 21 relevant studies; 14 were performed on mice, 5 on zebrafish and 2 on rats. We concluded that over forty-four genes in total are dispensable for fertility in both sexes without affecting host homeostasis. However, there are genes whose loss-of-function induces moderate to severe phenotypic changes in both sexes. There were situations in which the authors reported infertility, exhibited by the experimental model, or other pathologies such as cryptorchidism, cataracts, or reduced motor activity. Overall, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 are techniques that offer a wide range of possibilities for studying infertility, even to create mutant variants. It can be concluded that ZFNs, TALENs, and CRISPR/Cas9 are crucial tools in biomedical research.

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