scholarly journals Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

2019 ◽  
Vol 20 (15) ◽  
pp. 3719 ◽  
Author(s):  
Zahra Hajiahmadi ◽  
Ali Movahedi ◽  
Hui Wei ◽  
Dawei Li ◽  
Yasin Orooji ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Short Palindromic Repeat ◽  
External Forces ◽  
Time And Energy ◽  
Reduced Time ◽  
Target Effects

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

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.

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

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.

Concepts of Molecular Plant Breeding and Genome Editing

2021 ◽  
pp. 1-15
Keyword(s):  
Plant Breeding ◽  
Genome Editing ◽  
Genetic Linkage ◽  
Selection Process ◽  
Crop Improvement ◽  
Zinc Finger Nucleases ◽  
Induced Mutations ◽  
Transcription Activator ◽  
Targeted Gene Editing ◽  
Breeding Techniques

Traditional plant breeding depends on spontaneous and induced mutations available in the crop plants. Such mutations are rare and occur randomly. By contrast, molecular breeding and genome editing are advanced breeding techniques that can enhance the selection process and produce precisely targeted modifications in any crop. Identification of molecular markers, based on SSRs and SNPs, and the availability of high-throughput (HTP) genotyping platforms have accelerated the process of generating dense genetic linkage maps and thereby enhanced application of marker-assisted breeding for crop improvement. Advanced molecular biology techniques that facilitate precise, efficient, and targeted modifications at genomic loci are termed as “genome editing.” The genome editing tools include “zinc-finger nucleases (ZNFs),” “transcription activator-like effector nucleases (TALENs),” oligonucleotide-directed mutagenesis (ODM), and “clustered regularly interspersed short palindromic repeats (CRISPER/Cas) system,” which can be used for targeted gene editing. Concepts of molecular plant breeding and genome editing systems are presented in this chapter.

TALEN-mediated genome editing: prospects and perspectives

2014 ◽  
Vol 462 (1) ◽  
pp. 15-24 ◽  
Author(s):  
David A. Wright ◽  
Ting Li ◽  
Bing Yang ◽  
Martin H. Spalding
Keyword(s):  
Genome Editing ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Natural Processes ◽  
Current State ◽  
A Genome ◽  
Dna Dsbs ◽  
Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

scholarly journals kRISP-meR: A Reference-free Guide-RNA Design Tool for CRISPR/Cas9

2019 ◽  
Author(s):  
Mahmudur Rahman Hera ◽  
Amatur Rahman ◽  
Atif Rahman
Keyword(s):  
Genome Editing ◽  
Design Tool ◽  
Target Region ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Design ◽  
Reference Genomes ◽  
Target Effects

AbstractGenome editing using the CRISPR/Cas9 system requires designing guide RNAs (sgRNA) that are efficient and specific. Guide RNAs are usually designed using reference genomes which limits their use in organisms with no or incomplete reference genomes. Here, we present kRISP-meR, a reference free method to design sgRNAs for CRISPR/Cas9 system. kRISP-meR takes as input a target region and sequenced reads from the organism to be edited and generates sgRNAs that are likely to minimize off-target effects. Our analysis indicates that kRISP-meR is able to identify majority of the guides identified by a widely used sgRNA designing tool, without any knowledge of the reference, while retaining specificity.

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 Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects

2021 ◽  
Vol 12 ◽  
Author(s):  
Yufan Xu ◽  
Xiaorong Peng ◽  
Yanghao Zheng ◽  
Changzhong Jin ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Host Cells ◽  
Preliminary Evaluation ◽  
Major Barrier ◽  
Editing Efficiency ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Target Effects

Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.

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