scholarly journals Common computational tools for analyzing CRISPR screens

2021 ◽  
Author(s):  
Medina Colic ◽  
Traver Hart
Keyword(s):  
Drug Resistance ◽  
Genome Editing ◽  
Analytical Methods ◽  
Computational Analysis ◽  
Synthetic Lethality ◽  
Genetic Interactions ◽  
Computational Tools ◽  
Genome Regulation ◽  
Rna Targeting ◽  
State Of Art

CRISPR–Cas technology offers a versatile toolbox for genome editing, with applications in various cancer-related fields such as functional genomics, immunotherapy, synthetic lethality and drug resistance, metastasis, genome regulation, chromatic accessibility and RNA-targeting. The variety of screening platforms and questions in which they are used have caused the development of a wide array of analytical methods for CRISPR analysis. In this review, we focus on the algorithms and frameworks used in the computational analysis of pooled CRISPR knockout (KO) screens and highlight some of the most significant target discoveries made using these methods. Lastly, we offer perspectives on the design and analysis of state-of-art multiplex screening for genetic interactions.

Nicotine Workshop

1976 ◽  
Vol 8 (5) ◽  
Author(s):  
xxx
Keyword(s):  
Present State ◽  
Analytical Methods ◽  
Body Fluids ◽  
Advisory Board ◽  
Tobacco Company ◽  
Human Situation ◽  
Tobacco Alkaloids ◽  
Related Compounds ◽  
State Of Art

AbstractA workshop on problems related to the analysis of nicotine and nicotine metabolites in body fluids at levels pertinent to the human situation was held in November 1974 in Stockholm. It was organized by C. Enzell, B. Holmstedt and A. Pilotti at the request of the Medical Advisory Board of the Swedish Tobacco Company. The goal of the workshop was to summarize the present state of art in the area outlined by the organizers and to discuss critically the advantages and limitations of the different analytical methods available today. EIeven experts in the field of metabolism, detection and biosynthesis of nicotine and related compounds were therefore invited to present papers on these topics and to participate in the discussions. AIl speakers invited were able to attend and the papers were arranged in the following groups:Each speaker had one hour and a half at his disposal which included the discussion which, due to the informal atmosphere and the smaII number of participants, was very lively and fruitful. The papers read at this workshop comprise a very valuable coverage of recent research in the fields of metabolism of nicotine and minor tobacco alkaloids, and of the various methods available for detection of these alkaloids. The abstracts are given below, while full papers, now edited by A. Pilotti, can be obtained on request from C. Enzell of the Swedish Tobacco Company

scholarly journals Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

2008 ◽  
Vol 190 (17) ◽  
pp. 5841-5854 ◽  
Author(s):  
Helen Ting ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Metabolic Pathways ◽  
Synthetic Lethality ◽  
Nucleotide Metabolism ◽  
Genetic Interactions ◽  
Single Defect ◽  
Damage Repair ◽  
Mutant Combination ◽  
Dna Incorporation

ABSTRACT Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The dut-1 defect in Escherichia coli inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(dut) mutations]. According to the redundancy logic, most of these SL(dut) mutations should affect nucleotide metabolism. After a systematic search for SL(dut) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (tdk), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with dut are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (ung, polA, and xthA); (ii) double-strand DNA break repair (recA, recBC, and ruvABC); and (iii) chromosomal-dimer resolution (xerC, xerD, and ftsK). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal “defect-damage-repair” cycles linking unrelated metabolic pathways. In addition, two SL(dut) inserts (phoU and degP) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the “compensation” explanation for this synthetic lethality. We conclude that genetic interactions with dut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.

scholarly journals Mapping genetic interactions in cancer: a road to rational combination therapies

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Beril Tutuncuoglu ◽  
Nevan J. Krogan
Keyword(s):  
Anticancer Drugs ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Parp Inhibitors ◽  
Genetic Interactions ◽  
Parp Inhibition ◽  
Functional Dependencies ◽  
Combination Therapies ◽  
Systematic Screening ◽  
Rational Combination

Abstract The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies.

scholarly journals Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Kyunghun Min ◽  
Yuichi Ichikawa ◽  
Carol A. Woolford ◽  
Aaron P. Mitchell
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Drug Targets ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Content Type ◽  
Biological Features ◽  
Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9

Zebrafish ◽  
2016 ◽  
Vol 13 (1) ◽  
pp. 70-73 ◽  
Author(s):  
Sergey V. Prykhozhij ◽  
Vinothkumar Rajan ◽  
Jason N. Berman
Keyword(s):  
Genome Editing ◽  
Design Strategies ◽  
Computational Tools

scholarly journals Mad7: An IP Friendly CRISPR Enzyme

2021 ◽  
Author(s):  
Johan Rojek ◽  
Yogesh Basavaraju ◽  
Saranya Nallapareddy ◽  
Roland Franz Baumgartner ◽  
Sanne Schoffelen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cho Cells ◽  
Computational Tools ◽  
Engineering Community

Abstract Mad7 is a CRISPR enzyme, similar to Cas9 and Cas12a, which is of great interest to industry and academia due to its permissive licensing agreement, which states that everyone is free to use it but not distribute it commercially. This paper demonstrates that Mad7 genome editing works in CHO cells and provides the CHO engineering community with the computational tools needed to generate Mad7 compatible gRNAs against the CHO genome.

scholarly journals Integrated design, execution, and analysis of arrayed and pooled CRISPR genome editing experiments

2017 ◽  
Author(s):  
Matthew C. Canver ◽  
Maximilian Haeussler ◽  
Daniel E. Bauer ◽  
Stuart H. Orkin ◽  
Neville E. Sanjana ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput Sequencing ◽  
Integrated Design ◽  
Sequencing Data ◽  
Computational Tools ◽  
Guide Rnas ◽  
High Throughput Sequencing Data ◽  
Experimental Implementation ◽  
Computational Data ◽  
Sgrna Design

ABSTRACTCRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

scholarly journals Improved computational analysis of ribosome dynamics from 5’P degradome data using fivepeseq

2020 ◽  
Author(s):  
Lilit Nersisyan ◽  
Maria Ropat ◽  
Vicent Pelechano
Keyword(s):  
Computational Analysis ◽  
Developmental Stages ◽  
Biological Information ◽  
List Type ◽  
Computational Tools ◽  
Ribosome Stalling ◽  
Specific Analysis ◽  
Key Points ◽  
Reproducible Analysis

ABSTRACTIn eukaryotes, 5’-3’ co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus 5’P degradome sequencing, in addition to informing about RNA decay, also provides valuable information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome, however computational tools for their reproducible analysis are lacking. Here we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5’P degradome data. This tool performs both metagene and gene specific analysis, and allows to easily investigate codon specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene specific ribosome pauses in S. cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific frameshifts. To show its wide applicability, we investigate more complex 5’P degradome from A. thaliana and discover both motif-specific ribosome protection associated with particular developmental stages, as well as generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5’P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis.KEY POINTSAnalysis of 5’P degradome data with fivepseq informs about global and gene-specific translational features.Frameshifts in translation-related genes in S. cerevisiae may be linked to ribosome stalling.Ribosome protection at termination and codon motifs are linked to development in A. Thaliana.

scholarly journals Su1046 – Crispr/Cas9-Mediated Genome Editing of Multi-Drug Resistance Proteins in a Novel Canine Enteroid Model

2019 ◽  
Vol 156 (6) ◽  
pp. S-494
Author(s):  
Dana C. Borcherding ◽  
Yoko M. Ambrosini ◽  
Todd Atherly ◽  
Tori Rudolph ◽  
Jeffrey Essner ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Genome Editing ◽  
Multi Drug Resistance ◽  
Resistance Proteins

scholarly journals Computational Methods for Single-Cell RNA Sequencing

2020 ◽  
Vol 3 (1) ◽  
pp. 339-364 ◽  
Author(s):  
Brian Hie ◽  
Joshua Peters ◽  
Sarah K. Nyquist ◽  
Alex K. Shalek ◽  
Bonnie Berger ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Computational Methods ◽  
Analytical Methods ◽  
High Dimensional ◽  
Computational Tools ◽  
Single Cell Rna Sequencing ◽  
Selection Of

Single-cell RNA sequencing (scRNA-seq) has provided a high-dimensional catalog of millions of cells across species and diseases. These data have spurred the development of hundreds of computational tools to derive novel biological insights. Here, we outline the components of scRNA-seq analytical pipelines and the computational methods that underlie these steps. We describe available methods, highlight well-executed benchmarking studies, and identify opportunities for additional benchmarking studies and computational methods. As the biochemical approaches for single-cell omics advance, we propose coupled development of robust analytical pipelines suited for the challenges that new data present and principled selection of analytical methods that are suited for the biological questions to be addressed.

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