CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering

Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.

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Various Aspects of a Gene Editing System—CRISPR–Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms21249604 ◽

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.

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Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

Nucleic Acids Research ◽

10.1093/nar/gkt1074 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2577-2590 ◽

Cited By ~ 217

Author(s):

Ines Fonfara ◽

Anaïs Le Rhun ◽

Krzysztof Chylinski ◽

Kira S. Makarova ◽

Anne-Laure Lécrivain ◽

...

Keyword(s):

Genome Editing ◽

Horizontal Transfer ◽

Genome Engineering ◽

Bacterial Species ◽

Type Ii ◽

Promising Technology ◽

Dna Targeting ◽

Protospacer Adjacent Motif ◽

Cas9 Protein ◽

Specific Manner

Abstract The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

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CRISPR/CAS9 IN GENOME EDITING: A NATURE GIFTED MOLECULAR TOOL

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

10.54112/bcsrj.v2020i1.18 ◽

2020 ◽

Vol 2020 (1) ◽

Author(s):

MA Bashir ◽

Q Ali ◽

MS Rashid ◽

A Malika

Keyword(s):

Immune System ◽

Dna Sequence ◽

Genomic Dna ◽

Life Forms ◽

Type Ii ◽

Rna Sequences ◽

Genomic Engineering ◽

Genetic Tool ◽

Cas9 Protein ◽

Dna Editing

The Cas9 protein derived from type II CRISPR as a part of bacterial immune system has been raising up as a useful genetic tool for genomic engineering in various life forms. As RNA-guided DNA endonucleases, the Cas9 could be effectively customized to marked new DNA sequence sites by adjusting guided RNA sequences; it has been appeared as new emerging DNA editing technology. The nuclease-disable types of Cas9 has provided adaptable RNA guided DNA focusing on regulation and visualization of genomic DNA, just as for restoring the epigenetic forms and status, all has been shown in a an accurate sequence. Through these proceed; the researchers have started to explore conceivable uses of Cas9 in medical, agriculture, pharmaceutical and livestock sciences.

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Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells

Chemical Communications ◽

10.1039/c4cc02804j ◽

2014 ◽

Vol 50 (94) ◽

pp. 14724-14727 ◽

Cited By ~ 20

Author(s):

Ming Cheng ◽

Wei Zhang ◽

Jinghe Yuan ◽

Wangxi Luo ◽

Nan Li ◽

...

Keyword(s):

Growth Factor ◽

Single Molecule ◽

Transforming Growth Factor ◽

Living Cells ◽

Unnatural Amino Acid ◽

Type Ii ◽

Site Specific ◽

Single Molecule Dynamics ◽

Factor Type ◽

Molecule Dynamics

Single-molecule dynamics of the transforming growth factor type II receptor (TβRII) labeled by an unnatural amino acid.

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Emulators as a Tool for Control Digital System Software Debugging

INFORMACIONNYE TEHNOLOGII ◽

10.17587/it.27.339-349 ◽

2021 ◽

Vol 27 (7) ◽

pp. 339-349

Author(s):

A. D. Ivannikov ◽

Keyword(s):

Graphical Model ◽

High Efficiency ◽

Direct Memory Access ◽

Practical Method ◽

Digital System ◽

Digital Systems ◽

System Software ◽

System A ◽

Software Modules ◽

Operational Program

The simulation of control digital systems at the architecture level, that is, emulation of the instruction set, memory cells and internal programmable registers, as well as the interrupt system and direct memory access is considered. Emulators are used for debugging embedded digital system software and in the development of new custom processors. Requirements for emulators are formalized. It is shown that the main requirements are adequate simulation of digital systems at the architecture level, the presence of a set of convenient debugging modes, as well as high efficiency of emulators, that is, the minimum possible average number of instrumental computer instructions required to simulate one instruction of the target digital system. A classification of the debugging capabilities of emulators and possible ways of implementing debugging modes is given. The composition of the emulators is described. A graphical model of the structure of the emulator is proposed. The simulation process for each instruction is presented as a sequence of execution of smaller operations. If different instructions include the same operations, these operations can be performed by the same software modules. These modules can be included in all the corresponding blocks of the emulator that simulate the execution of instructions, or the emulator can include only one copy of each operational program module, and the module can be accessed while simulating the corresponding instruction. Determination of the structure of the emulator is formalized as an extreme task, the objective function of which is the minimum average time for simulating the execution of one instruction of the target digital system, and the limitation is the maximum allowable memory size of the instrumental computer occupied by the emulator. A practical method for determining the structure of the emulator is proposed.

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Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering

Genome ◽

10.1139/gen-2016-0127 ◽

2017 ◽

Vol 60 (6) ◽

pp. 537-545 ◽

Cited By ~ 6

Author(s):

Kumiko Ui-Tei ◽

Shohei Maruyama ◽

Yuko Nakano

Keyword(s):

Rna Polymerase Iii ◽

Protein A ◽

Genomic Region ◽

Guide Rna ◽

Genomic Engineering ◽

System A ◽

Double Stranded Dna ◽

Transcriptional Termination ◽

Sequence Complementarity ◽

Pol Iii

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

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Improved High-Efficiency Conventional Solar Photovoltaic System: A Novel Approach

Solar Power and Energy Storage Systems ◽

10.1201/9780429458774-2 ◽

2019 ◽

pp. 25-68

Author(s):

Hee-Je Kim

Keyword(s):

High Efficiency ◽

Photovoltaic System ◽

Solar Photovoltaic ◽

System A ◽

Novel Approach ◽

Solar Photovoltaic System

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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

10.20944/preprints202007.0578.v1 ◽

2020 ◽

Author(s):

Anindya Bandyopadhyay ◽

Nagesh Kancharla ◽

vivek javalkote ◽

santanu dasgupta ◽

Thomas Brutnell

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Temperature Stability ◽

Double Strand Breaks ◽

Plant Genome ◽

Strand Breaks ◽

Guide Rna ◽

Versatile Tool ◽

Type V ◽

Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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