Various Aspects of a Gene Editing System—CRISPR–Cas9

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20153719 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3719 ◽

Cited By ~ 14

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.

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CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

Cells ◽

10.3390/cells9112518 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2518

Author(s):

Maryam Saifaldeen ◽

Dana E. Al-Ansari ◽

Dindial Ramotar ◽

Mustapha Aouida

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

Catalytic Domain ◽

Zinc Finger Nucleases ◽

Dna Double Strand Breaks ◽

Transcription Activator ◽

Strand Breaks ◽

Protospacer Adjacent Motif ◽

Cas9 Protein ◽

Wide Range

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

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.

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Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

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CRISPR as a Versatile Technology for Gene Activation and Genome Editing

Journal of Genes and Cells ◽

10.15562/gnc.62 ◽

2018 ◽

Vol 4 ◽

pp. 5

Author(s):

Habib Rezanejadbardeji ◽

Bahareh Behroozi-Asl ◽

Raheleh Amirkhah

Keyword(s):

Immune System ◽

Therapeutic Potential ◽

Genetic Disorders ◽

Gene Activation ◽

Adaptive Immune System ◽

Stem Cell Differentiation ◽

Zinc Finger Nucleases ◽

Viral Gene ◽

Transcription Activator ◽

Adaptive Immune

CRISPR/Cas system, a microbial adaptive immune system, has rapidly transformed the ways researchers can interrogate the genome. CRISPR has many advantages over traditional methods such as Transcription activator-like effector nucleases (TALEN) and Zinc-finger nucleases (ZFN). Since CRISPR discovery as an adaptive immune system used by bacterial against viruses, it has been repurposed to help in many different genome-related studies such as gene knocking in and out, gene expression upregulation and downregulation. Also CRISPR holds vast therapeutic potential for the management of genetic disorders by straight modifying disease-causing mutations. Although the Cas9 protein has been revealed to attach and cleave DNA at off-target sites, the ﬁeld of Cas9 speciﬁcity is quickly progressing, with marked modifying in guide RNA choice, protein and guide engineering, innovative enzymes, and off-target recognition methods. In current review we mostly focus on CRISPR unique ability in gene activation/ upregulation, which has wide applications in different aspects such as gene studies, stem cell differentiation, and trans-differentiation. Compared to other gene activation methods such as viral gene overexpression, TALEN and ZFN, CRISPR offers many benefits such as easy designing and high precision.

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Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810062115 ◽

2018 ◽

Vol 115 (38) ◽

pp. 9351-9358 ◽

Cited By ~ 63

Author(s):

Robert M. Yarrington ◽

Surbhi Verma ◽

Shaina Schwartz ◽

Jonathan K. Trautman ◽

Dana Carroll

Keyword(s):

Genome Editing ◽

Zinc Finger Nucleases ◽

Yeast Genome ◽

Target Sequence ◽

Practical Applications ◽

Guide Rna ◽

Cas9 Protein ◽

Wide Range

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.

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A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement

International Journal of Molecular Sciences ◽

10.3390/ijms21165665 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5665 ◽

Cited By ~ 2

Author(s):

Sunny Ahmar ◽

Sumbul Saeed ◽

Muhammad Hafeez Ullah Khan ◽

Shahid Ullah Khan ◽

Freddy Mora-Poblete ◽

...

Keyword(s):

Genome Editing ◽

Transcriptional Control ◽

Gene Editing ◽

Genomic Structure ◽

Genetic Locus ◽

Crop Improvement ◽

Zinc Finger Nucleases ◽

Nucleotide Polymorphisms ◽

Transcription Activator ◽

Genetic Traits

Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems.

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Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

International Journal of Molecular Sciences ◽

10.3390/ijms20164045 ◽

2019 ◽

Vol 20 (16) ◽

pp. 4045 ◽

Cited By ~ 14

Author(s):

Ali Razzaq ◽

Fozia Saleem ◽

Mehak Kanwal ◽

Ghulam Mustafa ◽

Sumaira Yousaf ◽

...

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Crop Improvement ◽

Crop Plants ◽

Targeted Mutagenesis ◽

Plant Genome ◽

Zinc Finger Nucleases ◽

Base Substitution ◽

Transcription Activator ◽

Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

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Recent advances of Cas12a applications in bacteria

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11243-9 ◽

2021 ◽

Author(s):

Meliawati Meliawati ◽

Christoph Schilling ◽

Jochen Schmid

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Bacterial Genome ◽

Adaptive Immune System ◽

Eukaryotic Cell ◽

Promising Alternative ◽

Guide Rna ◽

Recent Advances ◽

Versatile Tool

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

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THE JOURNEY OF CRISPR-CAS9 FROM BACTERIAL DEFENSE MECHANISM TO A GENE EDITING TOOL IN BOTH ANIMALS AND PLANTS

Journal of Humanities, Social and Management Sciences (JHSMS) ◽

10.54112/bcsrj.v2020i1.17 ◽

2020 ◽

Vol 2020 (1) ◽

Author(s):

T Tahir ◽

Q Ali ◽

MS Rashid ◽

A Malik

Keyword(s):

Immune System ◽

Genetic Engineering ◽

Success Rate ◽

Genome Editing ◽

Zinc Finger ◽

Gene Editing ◽

Defense Mechanism ◽

Zinc Finger Nucleases ◽

Transcription Activator

Today we can use multiple of endonucleases for genome editing which has become very important and used in number of applications. We use sequence specific molecular scissors out of which, most important are mega nucleases, zinc finger nucleases, TALENS (Transcription Activator Like-Effector Nucleases) and CRISPR-Cas9 which is currently the most famous due to a number of reasons, they are cheap, easy to build, very specific in nature and their success rate in plants and animals is also high. Who knew that one day these CRISPR discovered as a part of immune system of bacteria will be this much worthwhile in the field of genetic engineering? This review interprets the science behind their mechanism and how several advancements were made with the passage of time to make them more efficient for the assigned job.

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