scholarly journals CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Krzysztof Chylinski ◽  
Maria Hubmann ◽  
Ruth E. Hanna ◽  
Connor Yanchus ◽  
Georg Michlits ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genome Engineering ◽  
Expression Cassette ◽  
Rapid Induction ◽  
Versatile Tool ◽  
Cre Recombination ◽  
Rapid Switching ◽  
Target Effects

AbstractCRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.

scholarly journals Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

2021 ◽  
Author(s):  
Nghi Dang ◽  
Alissa Lance-Byrne ◽  
Angela Tung ◽  
Xiaoge Guo ◽  
Ryan J Cecchi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Transient Transfection ◽  
Cell Engineering ◽  
Genetic Manipulations ◽  
Expression Plasmids

Abstract CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

scholarly journals Conditional control of RNA-guided nucleic acid cleavage and gene editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shao-Ru Wang ◽  
Ling-Yu Wu ◽  
Hai-Yan Huang ◽  
Wei Xiong ◽  
Jian Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Gene Editing ◽  
Chemical Activation ◽  
Live Cells ◽  
Guide Rna ◽  
Dna And Rna ◽  
Conditional Control ◽  
Acid Cleavage ◽  
Versatile Tool ◽  
Target Effects

AbstractProkaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.

CRISPR-Cas9 genome engineering: trends in medicine and health

2021 ◽  
Vol 21 ◽  
Author(s):  
Sumera Zaib ◽  
Mushtaq Saleem ◽  
Imtiaz Khan
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Genetic Manipulation ◽  
Engineering Process ◽  
High Cholesterol ◽  
The Past ◽  
Current Review ◽  
Medical Researchers ◽  
Short Palindromic Repeat ◽  
Target Effects

: The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine and biotechnology. Over the past few decades, the development of CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the whole genetic engineering process utilizing the principles of Watson-Crick base pairing. CRISPR-Cas9 technology offers the simplest, fastest, most versatile, reliable and precise method of genetic manipulation thus enabling geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. The current review focuses on the applications of CRISPR-Cas9 in the field of medical research. Compared with other gene editing technologies, CRISPR/Cas9 demonstrates numerous advantages for the treatment of various medical conditions including cancer, hepatitis B, cardiovascular diseases or even high cholesterol. Given its promising performance, CRISPR/Cas9 gene editing technology will surely help in the therapy of several disorders while addressing the issues pertaining to the minimization of the off-target effects of gene editing and incomplete matches between sgRNA and genomic DNA by Cas9.

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering

2019 ◽  
Vol 103 (11) ◽  
pp. 4313-4324 ◽  
Author(s):  
Ying Ding ◽  
Kai-Feng Wang ◽  
Wei-Jian Wang ◽  
Yi-Rong Ma ◽  
Tian-Qiong Shi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Recombination Efficiency ◽  
Eukaryotic Microorganisms

scholarly journals Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of “Rong and Golic”

2012 ◽  
Vol 21 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Rosalind Brookfield ◽  
Felix Dafhnis-Calas ◽  
Zhengyao Xu ◽  
William Brown
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Germ Line ◽  
Unsuccessful Attempt

scholarly journals Spatiotemporal control of CRISPR/Cas9 gene editing

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chenya Zhuo ◽  
Jiabin Zhang ◽  
Jung-Hwan Lee ◽  
Ju Jiao ◽  
Du Cheng ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Gene Editing ◽  
Genetic Regulation ◽  
Clinical Translation ◽  
Small Molecule Activation ◽  
Simple Design ◽  
Advantages And Disadvantages ◽  
Spatiotemporal Control ◽  
Critical Challenge ◽  
Target Effects

AbstractThe clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.

scholarly journals BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

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