scholarly journals An optimized base editor with efficient C-to-T base editing in zebrafish

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yu Zhao ◽  
Dantong Shang ◽  
Ruhong Ying ◽  
Hanhua Cheng ◽  
Rongjia Zhou
Keyword(s):  
Gene Editing ◽  
Cytidine Deaminase ◽  
Model Organism ◽  
Model Organisms ◽  
Base Substitution ◽  
Base Editing ◽  
Codon Preference ◽  
Promising Tool ◽  
Human Genes ◽  
Target Sites

Abstract Background Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Results Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Conclusions Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

scholarly journals Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

2020 ◽  
Author(s):  
Xi Xiang ◽  
Kunli Qu ◽  
Xue Liang ◽  
Xiaoguang Pan ◽  
Jun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
High Efficiency ◽  
Outcome Data ◽  
Massively Parallel ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Sites ◽  
In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

scholarly journals The Future of Gene-Guided Neuroscience Research in Non-Traditional Model Organisms

2019 ◽  
Vol 93 (2-3) ◽  
pp. 108-121 ◽  
Author(s):  
Scott Juntti
Keyword(s):  
Gene Editing ◽  
High Throughput Sequencing ◽  
Model Organism ◽  
Model Organisms ◽  
Traditional Model ◽  
Model Species ◽  
Neuroscience Research ◽  
Experimental Approaches ◽  
Natural Variations ◽  
And Behavior

Natural variations across animals in form, function, and behavior have long been sources of inspiration to scientists. Despite this, experimentalists focusing on the neural bases of behavior have increasingly focused on a select few model species. This consolidation is motivated primarily by the availability of resources and technologies for manipulation in these species. Recent years have witnessed a proliferation of experimental approaches that were developed primarily in traditional model species, but that may in principle be readily applied to any species. High-throughput sequencing, CRISPR gene editing, transgenesis, and other technologies have enabled new insights through their deployment in non-traditional model species. The availability of such approaches changes the calculation of which species to study, particularly when a trait of interest is most readily observed in a non-traditional model organism. If these technologies are widely adopted in many new species, it promises to revolutionize the field of neuroethology.

scholarly journals Comparative genomics within and across Bilaterians illuminates the evolutionary history of ALK and LTK proto-oncogene origination and diversification

2020 ◽  
Author(s):  
Alex Dornburg ◽  
Zheng Wang ◽  
Junrui Wang ◽  
Elizabeth S Mo ◽  
Francesc Lopez-Giraldez ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Model Organism ◽  
Large Cell ◽  
Model Organisms ◽  
Close Relative ◽  
Comparative Genomic ◽  
Protein Coding ◽  
Functional Roles ◽  
Anaplastic Lymphoma ◽  
Human Genes

Abstract Comparative genomic analyses have enormous potential for identifying key genes central to human health phenotypes, including those that promote cancers. In particular, the successful development of novel therapeutics using model species requires phylogenetic analyses to determine molecular homology. Accordingly, we investigate the evolutionary histories of anaplastic lymphoma kinase (ALK)—which can underlie tumorigenesis in neuroblastoma, non-small cell lung cancer, and anaplastic large-cell lymphoma—its close relative leukocyte tyrosine kinase (LTK) and their candidate ligands. Homology of ligands identified in model organisms to those functioning in humans remains unclear. Therefore, we searched for homologs of the human genes across metazoan genomes, finding that the candidate ligands Jeb and Hen-1 were restricted to non-vertebrate species. In contrast, the ligand AUG was only identified in vertebrates. We found two ALK-like and four AUG-like protein-coding genes in lamprey. Of these six genes, only one ALK-like and two AUG-like genes exhibited early embryonic expression that parallels model mammal systems. Two copies of AUG are present in nearly all jawed vertebrates. Our phylogenetic analysis strongly supports the presence of previously unrecognized functional convergences of ALK and LTK between actinopterygians and sarcopterygians—despite contemporaneous, highly conserved synteny of ALK and LTK. These findings provide critical guidance regarding the propriety of fish and mammal models with regard to model-organism-based investigation of these medically important genes. In sum, our results provide the phylogenetic context necessary for effective investigations of the functional roles and biology of these critically important receptors.

scholarly journals High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity

2021 ◽  
Vol 22 (24) ◽  
pp. 13301
Author(s):  
Dan Zhu ◽  
Junyi Wang ◽  
Di Yang ◽  
Jianzhong Xi ◽  
Juan Li
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
Genome Engineering ◽  
Human Cells ◽  
Wild Type ◽  
Guide Rnas ◽  
Promising Tool ◽  
Target Sites ◽  
Editing Activity ◽  
Type V

CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position −3 for PAM TATV and at position −4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.

scholarly journals The past, present and future of Dictyostelium as a model system

2019 ◽  
Vol 63 (8-9-10) ◽  
pp. 321-331 ◽  
Author(s):  
Salvatore Bozzaro
Keyword(s):  
Dictyostelium Discoideum ◽  
Social Evolution ◽  
Model Organism ◽  
Model Organisms ◽  
Human Genes ◽  
The Social ◽  
Microbial Infections ◽  
Technological Developments ◽  
Soil Amoeba

The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis, phagocytosis and macropinocytosis, intercellular adhesion, pattern formation, caspase-independent cell death and more recently autophagy and social evolution. Being a soil amoeba and professional phagocyte, thus exposed to a variety of potential pathogens, D. discoideum has also proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections. The finding that the Dictyostelium genome harbours several homologs of human genes responsible for a variety of diseases has stimulated their analysis, providing new insights into the mechanism of action of the encoded proteins and in some cases into the defect underlying the disease. Recent technological developments have covered the genetic gap between mammals and non-mammalian model organisms, challenging the modelling role of the latter. Is there a future for Dictyostelium discoideum as a model organism?

scholarly journals A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

2019 ◽  
Author(s):  
Rina C. Sakata ◽  
Soh Ishiguro ◽  
Hideto Mori ◽  
Mamoru Tanaka ◽  
Motoaki Seki ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cytidine Deaminase ◽  
Sequencing Data ◽  
Base Editing ◽  
Dna Molecule ◽  
Deep Sequencing Data ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites ◽  
Sequence Window

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G mutation at target genomic sites, the possible genome editing space when using the current base editors remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of Target-ACE and other base editors were simulated across the human genome, demonstrating the highest potency of Target-ACE to edit amino acid coding patterns. Taking these findings together, Target-ACE is a new tool that broadens the capabilities for base editing for various applications.

CRISPR/Cas9 Genome Editing Tool: A Promising Tool for Therapeutic Applications on Respiratory Diseases

2020 ◽  
Vol 20 (5) ◽  
pp. 333-346
Author(s):  
Sadiya Bi Shaikh ◽  
Yashodhar Prabhakar Bhandary
Keyword(s):  
Respiratory Diseases ◽  
Gene Editing ◽  
Epithelial Mesenchymal Transition ◽  
Respiratory Health ◽  
Chronic Obstructive ◽  
Therapeutic Tool ◽  
Obstructive Pulmonary Disease ◽  
Mesenchymal Transition ◽  
Chronic Respiratory Diseases ◽  
Promising Tool

Respiratory diseases are one of the prime topics of concern in the current era due to improper diagnostics tools. Gene-editing therapy, like Clustered regularly interspaced palindromic repeats- associated nuclease 9 (CRISPR/Cas9), is gaining popularity in pulmonary research, opening up doors to invaluable insights on underlying mechanisms. CRISPR/Cas9 can be considered as a potential gene-editing tool with a scientific community that is helping in the advancement of knowledge in respiratory health and therapy. As an appealing therapeutic tool, we hereby explore the advanced research on the application of CRISPR/Cas9 tools in chronic respiratory diseases such as lung cancer, Acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF). We also address the urgent need to establish this gene-editing tool in various other lung diseases such as asthma, Chronic obstructive pulmonary disease (COPD) and Idiopathic pulmonary fibrosis (IPF). The present review introduces CRISPR/Cas9 as a worthy application in targeting epithelial-mesenchymal transition and fibrinolytic system via editing specific genes. Thereby, based on the efficiency of CRISPR/Cas9, it can be considered as a promising therapeutic tool in respiratory health research.

scholarly journals In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))—Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2226
Author(s):  
Sazia Kunvar ◽  
Sylwia Czarnomska ◽  
Cino Pertoldi ◽  
Małgorzata Tokarska
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Bos Taurus ◽  
Model Organism ◽  
Genotyping By Sequencing ◽  
Model Organisms ◽  
European Bison ◽  
Model Animal ◽  
Pcr Duplicates ◽  
Species Specific

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

scholarly journals New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

2020 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
Jaiana Malabarba ◽  
Elisabeth Chevreau ◽  
Nicolas Dousset ◽  
Florian Veillet ◽  
Julie Moizan ◽  
...  
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Acetolactate Synthase ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Adventitious Regeneration ◽  
Base Editing ◽  
Perennial Plants ◽  
Guide Rnas ◽  
Albino Phenotype

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

scholarly journals Alliance of Genome Resources Portal: unified model organism research platform

2019 ◽  
Vol 48 (D1) ◽  
pp. D650-D658 ◽  
Author(s):  
◽  
Julie Agapite ◽  
Laurent-Philippe Albou ◽  
Suzi Aleksander ◽  
Joanna Argasinska ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Model Organism ◽  
Model Organisms ◽  
Data Types ◽  
Primary Model ◽  
Genomic Studies ◽  
Health And Disease ◽  
Extensive Body ◽  
Access To Data ◽  
Model Organism Databases

Abstract The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource.

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