scholarly journals Highly efficient CRISPR gene editing in yeast enabled by double selection

2018 ◽  
Author(s):  
Philippe C Després ◽  
Alexandre K Dubé ◽  
Lou Nielly-Thibault ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
High Frequency ◽  
Large Scale ◽  
Gene Editing ◽  
Chromosomal Translocations ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Base Editing ◽  
Fitness Effects ◽  
Genetics And Genomics ◽  
Selection For

AbstractCRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

scholarly journals Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

2020 ◽  
Vol 10 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Function ◽  
Good Predictor ◽  
Gene Editing ◽  
Selection Strategy ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Non Coding Rnas

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

scholarly journals Universal toxin-based selection for precise genome engineering in human cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Songyuan Li ◽  
Nina Akrap ◽  
Silvia Cerboni ◽  
Michelle J. Porritt ◽  
Sandra Wimberger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
Genetically Engineered ◽  
Human Cells ◽  
Selection Strategy ◽  
Nucleotide Substitutions ◽  
Base Editing ◽  
Selection For

AbstractProkaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

scholarly journals Review: Balancing Selection for Deleterious Alleles in Livestock

2021 ◽  
Vol 12 ◽  
Author(s):  
Martijn F. L. Derks ◽  
Marije Steensma
Keyword(s):  
Balancing Selection ◽  
Low Frequency ◽  
Purifying Selection ◽  
Population Level ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Deleterious Alleles ◽  
Functional Consequences ◽  
Selection For ◽  
The Impact

Harmful alleles can be under balancing selection due to an interplay of artificial selection for the variant in heterozygotes and purifying selection against the variant in homozygotes. These pleiotropic variants can remain at moderate to high frequency expressing an advantage for favorable traits in heterozygotes, while harmful in homozygotes. The impact on the population and selection strength depends on the consequence of the variant both in heterozygotes and homozygotes. The deleterious phenotype expressed in homozygotes can range from early lethality to a slightly lower fitness in the population. In this review, we explore a range of causative variants under balancing selection including loss-of-function variation (i.e., frameshift, stop-gained variants) and regulatory variation (affecting gene expression). We report that harmful alleles often affect orthologous genes in different species, often influencing analogous traits. The recent discoveries are mainly driven by the increasing genomic and phenotypic resources in livestock populations. However, the low frequency and sometimes subtle effects in homozygotes prevent accurate mapping of such pleiotropic variants, which requires novel strategies to discover. After discovery, the selection strategy for deleterious variants under balancing selection is under debate, as variants can contribute to the heterosis effect in crossbred animals in various livestock species, compensating for the loss in purebred animals. Nevertheless, gene-assisted selection is a useful tool to decrease the frequency of the harmful allele in the population, if desired. Together, this review marks various deleterious variants under balancing selection and describing the functional consequences at the molecular, phenotypic, and population level, providing a resource for further study.

scholarly journals Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

2019 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Gene Function ◽  
Gene Editing ◽  
Gene Families ◽  
Single Copy ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Polyploid Plant ◽  
Non Coding Rnas

AbstractCRISPR-Cas9 has become the preferred gene editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprograming of Cas9 specificity using customizable small non-coding RNAs, and to the ability to target several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource consuming. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function mutation produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxies. We found all three proxies predicted the occurrence of mutations in either or both of the other two proxies with efficiencies ranging from 40% to 100%, dramatically reducing the number of plants that need to be screened to identify CRISPR mutants. This selection strategy provides a framework to facilitate gene function studies of gene families as well as the function of single copy genes in polyploid plant species where the identification of multiplex mutants remains challenging.

Kindler's Syndrome with Recurrent Neutropenia: Report of Two Cases from Saudi Arabia

2020 ◽  
Author(s):  
Yousef Binamer ◽  
Muzamil A. Chisti
Keyword(s):  
Autosomal Recessive ◽  
Common Mechanism ◽  
Sequencing Analysis ◽  
Loss Of Function ◽  
Skin Atrophy ◽  
Whole Exome ◽  
Infancy And Childhood ◽  
Genetics And Genomics ◽  
New Feature ◽  
Generation Sequencing

AbstractKindler syndrome (KS) is a rare photosensitivity disorder with autosomal recessive mode of inheritance. It is characterized by acral blistering in infancy and childhood, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility. Besides these major features, many minor presentations have also been reported in the literature. We are reporting two cases with atypical features of the syndrome and a new feature of recurrent neutropenia. Whole exome sequencing analysis was done using next-generation sequencing which detected a homozygous loss-of-function (LOF) variant of FERMT1 in both patients. The variant is classified as a pathogenic variant as per the American College of Medical Genetics and Genomics guidelines. Homozygous LOF variants of FERMT1 are a common mechanism of KS and as such confirm the diagnosis of KS in our patients even though the presentation was atypical.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals Optimizing the Frequency Capping: A Robust and Reliable Methodology to Define the Number of Ads to Maximize ROAS

2021 ◽  
Vol 11 (15) ◽  
pp. 6688
Author(s):  
Jesús Romero Leguina ◽  
Ángel Cuevas Rumin ◽  
Rubén Cuevas Rumin
Keyword(s):  
High Frequency ◽  
Large Scale ◽  
Low Frequency ◽  
Digital Marketing ◽  
Optimal Frequency ◽  
Novel Technique ◽  
Maximum Threshold

The goal of digital marketing is to connect advertisers with users that are interested in their products. This means serving ads to users, and it could lead to a user receiving hundreds of impressions of the same ad. Consequently, advertisers can define a maximum threshold to the number of impressions a user can receive, referred to as Frequency Cap. However, low frequency caps mean many users are not engaging with the advertiser. By contrast, with high frequency caps, users may receive many ads leading to annoyance and wasting budget. We build a robust and reliable methodology to define the number of ads that should be delivered to different users to maximize the ROAS and reduce the possibility that users get annoyed with the ads’ brand. The methodology uses a novel technique to find the optimal frequency capping based on the number of non-clicked impressions rather than the traditional number of received impressions. This methodology is validated using simulations and large-scale datasets obtained from real ad campaigns data. To sum up, our work proves that it is feasible to address the frequency capping optimization as a business problem, and we provide a framework that can be used to configure efficient frequency capping values.

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