scholarly journals Recent advances in structural studies of the CRISPR-Cas-mediated genome editing tools

2018 ◽  
Vol 6 (3) ◽  
pp. 438-451 ◽  
Author(s):  
Yuwei Zhu ◽  
Zhiwei Huang
Keyword(s):  
Nucleic Acids ◽  
Genome Editing ◽  
Adaptive Immunity ◽  
Structural Biology ◽  
Structural Studies ◽  
Guide Rnas ◽  
Recent Advances ◽  
Cas Proteins

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (Cas) proteins provide RNA-guided adaptive immunity for prokaryotes to defend themselves against viruses. The CRISPR-Cas systems have attracted much attention in recent years for their power in aiding the development of genome editing tools. Based on the composition of the CRISPR RNA-effector complex, the CRISPR-Cas systems can be divided into two classes and six types. In this review, we summarize recent advances in the structural biology of the CRISPR-Cas-mediated genome editing tools, which helps us to understand the mechanism of how the guide RNAs assemble with diverse Cas proteins to cleave target nucleic acids.

scholarly journals CRISPR-Cas System: The Powerful Modulator of Accessory Genomes in Prokaryotes

2021 ◽  
pp. 1-16
Author(s):  
Anca Butiuc-Keul ◽  
Anca Farkas ◽  
Rahela Carpa ◽  
Dumitrana Iordache
Keyword(s):  
Nucleic Acids ◽  
Pathogenic Bacteria ◽  
The Novel ◽  
Defense System ◽  
Guide Rnas ◽  
Crispr Array ◽  
Different Types ◽  
Evolutionary Trajectories ◽  
Novel Coronavirus ◽  
Cas Proteins

Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (<i>cas</i>)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system&apos;s impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus – SARS-CoV-2; thus, the newest and promising applications are reviewed as well.

scholarly journals Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Takuya Aoshima ◽  
Yukari Kobayashi ◽  
Hisayoshi Takagi ◽  
Kenta Iijima ◽  
Masahiro Sato ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Genome Editing ◽  
New Technology ◽  
Rat Fetuses ◽  
Guide Rnas ◽  
Culture Cells ◽  
Insertion And Deletion ◽  
Target Locus ◽  
Nucleic Acids Delivery

Abstract Background Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. Results The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. Conclusions We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

scholarly journals Re-purposing Ac/Ds transgenic system for CRISPR/dCas9 modulation of enhancers and non-coding RNAs in zebrafish

2018 ◽  
Author(s):  
Vanessa Chong-Morrison ◽  
Filipa C. Simões ◽  
Upeka Senanayake ◽  
Dervla S. Carroll ◽  
Paul R. Riley ◽  
...  
Keyword(s):  
Genome Editing ◽  
Critical Role ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Genetic Sequence ◽  
Guide Rnas ◽  
Excellent Model ◽  
Recent Advances ◽  
Summary Statement ◽  
Non Coding Rnas

AbstractDue to its genetic amenability coupled with recent advances in genome editing, the zebrafish serves as an excellent model to examine the function of both coding and non-coding elements. Recently, the non-coding genome has gained prominence due to its critical role in development and disease. Here, we have re-purposed the Ac/Ds maize transposition system to reliably screen and efficiently characterise zebrafish enhancers, with or without germline propagation. We further utilised the system to stably express guide RNAs in microinjected embryos enabling tissue-specific CRISPR/dCas9-interference (CRISPRi) knockdown of lncRNA and enhancer activity without disrupting the underlying genetic sequence. Our study highlights the utility of Ac/Ds transposition for transient epigenome modulation of non-coding elements in zebrafish.Summary statementWe adapted the Ac/Ds transposition system, which enables continuous expression of guide RNAs for CRISPR/dCas9 perturbation, to examine the function of non-coding RNAs and enhancer elements in zebrafish.

scholarly journals The basic building blocks and evolution of CRISPR–Cas systems

2013 ◽  
Vol 41 (6) ◽  
pp. 1392-1400 ◽  
Author(s):  
Kira S. Makarova ◽  
Yuri I. Wolf ◽  
Eugene V. Koonin
Keyword(s):  
Adaptive Immunity ◽  
Purifying Selection ◽  
Building Blocks ◽  
Comparative Sequence Analysis ◽  
Structural Rearrangements ◽  
Recognition Mechanism ◽  
Recent Advances ◽  
Self Recognition ◽  
First Time ◽  
Cas Proteins

CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) is an adaptive immunity system in bacteria and archaea that functions via a distinct self/non-self recognition mechanism that involves unique spacers homologous with viral or plasmid DNA and integrated into the CRISPR loci. Most of the Cas proteins evolve under relaxed purifying selection and some underwent dramatic structural rearrangements during evolution. In many cases, CRISPR–Cas system components are replaced either by homologous or by analogous proteins or domains in some bacterial and archaeal lineages. However, recent advances in comparative sequence analysis, structural studies and experimental data suggest that, despite this remarkable evolutionary plasticity, all CRISPR–Cas systems employ the same architectural and functional principles, and given the conservation of the principal building blocks, share a common ancestry. We review recent advances in the understanding of the evolution and organization of CRISPR–Cas systems. Among other developments, we describe for the first time a group of archaeal cas1 gene homologues that are not associated with CRISPR–Cas loci and are predicted to be involved in functions other than adaptive immunity.

scholarly journals Genome Editing in Bacteria: CRISPR-Cas and Beyond

2021 ◽  
Vol 9 (4) ◽  
pp. 844
Author(s):  
Ruben D. Arroyo-Olarte ◽  
Ricardo Bravo Rodríguez ◽  
Edgar Morales-Ríos
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Bacterial Genome ◽  
Guide Rnas ◽  
Comprehensive View ◽  
Ribonucleoprotein Complexes ◽  
Eukaryotic Organisms ◽  
Multi Step Methods ◽  
Cas Proteins

Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.

scholarly journals CRISPR interference: a structural perspective

2013 ◽  
Vol 453 (2) ◽  
pp. 155-166 ◽  
Author(s):  
Judith Reeks ◽  
James H. Naismith ◽  
Malcolm F. White
Keyword(s):  
Structural Biology ◽  
Recent Progress ◽  
Rna Binding ◽  
Protein Domain ◽  
Type I ◽  
Dependent Manner ◽  
Structural Studies ◽  
Future Studies ◽  
Associated Proteins ◽  
Cas Proteins

CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.

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.

Recent advances of genome editing and related technologies in China

Gene Therapy ◽  
2020 ◽  
Vol 27 (7-8) ◽  
pp. 312-320
Author(s):  
Wen Sun ◽  
Haoyi Wang
Keyword(s):  
Genome Editing ◽  
Recent Advances
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