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 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/Cas9-mediated genome editing in a reef-building coral

2018 ◽  
Vol 115 (20) ◽  
pp. 5235-5240 ◽  
Author(s):  
Phillip A. Cleves ◽  
Marie E. Strader ◽  
Line K. Bay ◽  
John R. Pringle ◽  
Mikhail V. Matz
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Mutant Gene ◽  
Mutation Induction ◽  
Induced Mutations ◽  
Important Species ◽  
Ribonucleoprotein Complexes ◽  
Genes Encoding ◽  
Green Fluorescent

Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals’ responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora. We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in ∼50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.

scholarly journals Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

2018 ◽  
Author(s):  
Fan Li ◽  
Sandy S.C. Hung ◽  
Jiang-Hui Wang ◽  
Vicki Chrysostomou ◽  
Vickie H.Y. Wong ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Genome Editing ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Extended Period ◽  
Widespread Application ◽  
Adeno Associated Virus ◽  
Safe Delivery ◽  
Guide Rnas

ABSTRACTSafe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application ofin vivogenome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing “kamikaze” CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to targetStreptococcus pyogenesCas9 (SpCas9), afterin situvalidation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 andThy1-YFPtransgenic mice. After 8 weeks the expression of SpCas9, the efficacy ofYFPgene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina ofThy1-YFPtransgenic mice. In conclusion, our data suggest that a self-destructive “kamikaze” CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.

scholarly journals Outcomes Comparison of SpCas9- and LbCas12a-mediated DNA Editing in Zebrafish Embryos

2020 ◽  
Author(s):  
Darya Meshalkina ◽  
Aleksei Glushchenko ◽  
Elana Kysil ◽  
Igor Mizgirev ◽  
Andrej Frolov
Keyword(s):  
Genome Editing ◽  
Mode Of Action ◽  
Target Gene ◽  
Gene Knockout ◽  
The Other ◽  
Zebrafish Embryos ◽  
Guide Rnas ◽  
Research Technology ◽  
Ribonucleoprotein Complexes ◽  
Dna Editing

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

scholarly journals Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

2018 ◽  
Author(s):  
Roswitha A. Aumann ◽  
Marc F. Schetelig ◽  
Irina Häecker
Keyword(s):  
Control Systems ◽  
Genome Editing ◽  
Pest Control ◽  
Genetically Modified Organisms ◽  
Ceratitis Capitata ◽  
Fluorescent Protein ◽  
Great Promise ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes

AbstractBackgroundThe Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic.ResultsHere we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring.ConclusionConsidering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

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 Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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