scholarly journals Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae

2019 ◽  
Vol 19 (7) ◽  
Author(s):  
Matthew Deaner ◽  
Hal S Alper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Gene Editing ◽  
Genome Engineering ◽  
Guide Rna ◽  
On Demand ◽  
Large Numbers ◽  
Genetic Modifications ◽  
Genome Scale

ABSTRACT Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this minireview, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting-edge techniques to explore gene editing and transcriptional regulation at genome scale using pooled approaches. The focus is on applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled single-guide RNA libraries and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes.

MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries

2019 ◽  
Author(s):  
Tao Li ◽  
Shaokai Wang ◽  
Feng Luo ◽  
Fang-Xiang Wu ◽  
Jianxin Wang
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Supplementary Information ◽  
Guide Rna ◽  
Multiple Processes ◽  
Design Guide ◽  
Computationally Expensive ◽  
Genomic Regions ◽  
Process Mode ◽  
Large Genomes

Abstract Summary The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9–12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes. Availability and implementation: MultiGuideScan is available at GitHub https://github.com/bioinfomaticsCSU/MultiGuideScan. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals The N-Terminal Tail of Histone H3 Regulates Copper Homeostasis in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Sakshi Singh ◽  
Rakesh Kumar Sahu ◽  
Raghuvir Singh Tomar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Histone H3 ◽  
Copper Homeostasis ◽  
Tata Binding Protein ◽  
Copper Stress ◽  
Toxic Level ◽  
Cellular Processes

Copper homeostasis is crucial for cellular processes. The balance between nutritional and toxic level is maintained through the regulation of uptake, distribution and detoxification via antagonistic actions of two transcription factors AceI and Mac1. AceI responds to toxic copper levels by transcriptional regulation of detoxification genes CUP1 and CRS5. Cup1 metallothionein (MT) confers protection against toxic copper levels. CUP1 gene regulation is a multifactorial event requiring AceI, TBP (TATA-binding protein), chromatin remodeler, acetyltransferase (Spt10) and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of H3 tail in CUP1 transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, Δ29-32) that reduce CUP1 expression. We detected reduced AceI occupancy across CUP1 promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28 and Δ28-31 correlating with the reduced CUP1 transcription. Majority of these mutations affect TBP occupancy at CUP1 promoter augmenting the CUP1 transcription defect. Additionally, some mutants display cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of H3 N-terminal tail and their modifications in CUP1 regulation.

scholarly journals Plant-based biosensors for detecting CRISPR-mediated genome engineering

2021 ◽  
Author(s):  
Guoliang Yuan ◽  
Md Mahmudul Hassan ◽  
Tao Yao ◽  
Haiwei Lu ◽  
Michael Melesse Vergara ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Transient Expression ◽  
Gene Editing ◽  
Genome Engineering ◽  
Protoplast Transformation ◽  
Reliable System ◽  
Base Editing ◽  
Detection Systems ◽  
Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica

2020 ◽  
Vol 9 (4) ◽  
pp. 967-971 ◽  
Author(s):  
Adithya Ramesh ◽  
Thomas Ong ◽  
Jaime A. Garcia ◽  
Jessica Adams ◽  
Ian Wheeldon
Keyword(s):  
Gene Regulation ◽  
Yarrowia Lipolytica ◽  
Gene Editing ◽  
Dual Purpose ◽  
Guide Rna

scholarly journals A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

2018 ◽  
Author(s):  
Zhanqi Dong ◽  
Qi Qin ◽  
Zhigang Hu ◽  
Peng Chen ◽  
Liang Huang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Sustained Delivery ◽  
Dna Breaks ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Transgenic Silkworms ◽  
Enzyme Targeting ◽  
Multiplex System ◽  
Unique Capability

Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

scholarly journals Multiplexed conditional genome editing with Cas12a in Drosophila

2020 ◽  
Vol 117 (37) ◽  
pp. 22890-22899 ◽  
Author(s):  
Fillip Port ◽  
Maja Starostecka ◽  
Michael Boutros
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
State Of The Art ◽  
Model Organism ◽  
Target Space ◽  
Guide Rna ◽  
Genome Modification ◽  
Conditional Expression ◽  
Targeted Genome Modification

CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila. We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.

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 Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Kunwoo Lee ◽  
Vanessa A Mackley ◽  
Anirudh Rao ◽  
Anthony T Chong ◽  
Mark A Dewitt ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Cell Sorting ◽  
Gene Editing ◽  
Genome Engineering ◽  
Cationic Polymers ◽  
Chemical Modifications ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Chemically Modified ◽  
New Strategies

Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5’ fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.

scholarly journals Transcription at a Distance in the Budding Yeast Saccharomyces cerevisiae

2021 ◽  
Vol 1 (1) ◽  
pp. 142-148
Author(s):  
JerryAnna Spiegel ◽  
James Arnone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Current Literature ◽  
Random Distribution ◽  
Budding Yeast ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Development

Proper transcriptional regulation depends on the collaboration of multiple layers of control simultaneously. Cells tightly balance cellular resources and integrate various signaling inputs to maintain homeostasis during growth, development and stressors, among other signals. Many eukaryotes, including the budding yeast Saccharomyces cerevisiae, exhibit a non-random distribution of functionally related genes throughout their genomes. This arrangement coordinates the transcription of genes that are found in clusters, and can occur over long distances. In this work, we review the current literature pertaining to gene regulation at a distance in budding yeast.

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