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 RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

2019 ◽  
Author(s):  
Adrian Kovač ◽  
Csaba Miskey ◽  
Michael Menzel ◽  
Esther Grueso ◽  
Andreas Gogol-Döring ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Sleeping Beauty ◽  
Specific Gene ◽  
Guide Rna ◽  
Gene Insertion ◽  
Genome Wide ◽  
Asymmetric Pattern ◽  
Integration Sites ◽  
Genomic Regions ◽  
Selection Of

ABSTRACTTwo different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, occurred in a relatively narrow, ∼200 bp window around the targeted sites and displayed an asymmetric pattern with a bias towards sites that are downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for efficient and specific gene insertion for precision genetic engineering.

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.

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 Design CRISPR-Cas9 Guide RNA and Donor Oligo to Introduce Pathogenic Mutation into Mice

2021 ◽  
Vol 2 (3) ◽  
Author(s):  
Xijing Liao ◽  
Xueyan Yang
Keyword(s):  
Medical Research ◽  
Cell Line ◽  
Gene Editing ◽  
Pathogenic Mutation ◽  
Genomic Sequences ◽  
Guide Rna ◽  
Pathogenic Effect ◽  
Design Guide

CRISPR-Ca9 system is a newly developed gene-editing technology, which is widely used in biology and medical research. In this project, we want to knock-in a mutation found in a human pedigree into mice through CRISPR-Cas9 technology to validate its pathogenic effect. We download corresponding mice genomic sequences and design guide RNA and donor oligo sequences according to CRISPR-Cas9 target principles. Following experiments confirm that this set of sequences is effective in mice cell line.

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 Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

2016 ◽  
Author(s):  
Wei Wang ◽  
Alina Akhunova ◽  
Shiaoman Chao ◽  
Eduard Akhunov
Keyword(s):  
Genome Editing ◽  
Transient Expression ◽  
Gene Editing ◽  
Genome Engineering ◽  
Agronomic Traits ◽  
Rna Polymerase Iii ◽  
Wheat Genome ◽  
Great Promise ◽  
Transient Expression Assay ◽  
Genomic Regions

AbstractBackgroundCRISPR/Cas9-based genome editing holds great promise to accelerate the development of new crop varieties by providing a powerful tool to modify the genomic regions controlling major agronomic traits. To diversify the set of tools available for wheat genome engineering, we have established a tRNA-based multiplex gene editing strategy for hexaploid wheat.ResultsThe functionality of the various CRISPR/Cas9 components was assessed using the transient expression in the wheat protoplasts followed by next-generation sequencing (NGS) of the targeted genomic regions. The efficiency of wheat codon-optimized Cas9 for targeted gene editing in wheat was validated. Multiple single guide RNAs (gRNAs) were evaluated for the ability to edit the homoeologous copies of four genes affecting some important agronomic traits in wheat. Low correspondence was found between the gRNA efficiency predicted bioinformatically and that assessed in the transient expression assay. A multiplex gene editing construct with several gRNA-tRNA units under the control of a single promoter for the RNA polymerase III generated indels at the targets sites with the efficiency comparable to that obtained for a single gRNA construct.ConclusionsBy integrating the protoplast transformation assay with multiplexed NGS, it is possible to perform fast functional screens for a large number of gRNAs and to optimize constructs for effective editing of multiple independent targets in the wheat genome. The multiplexing capacity of the tandemly arrayed tRNA–gRNA construct is well suited for the simultaneous editing of the redundant gene copies in the allopolyploid genomes or genomic regions beneficially affecting multiple agronomic traits. A polycistronic gene construct that can be quickly assembled using the Golden Gate reaction along with the wheat codon optimized Cas9 will further expand the set of tools available for engineering the wheat genome.

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 Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

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