scholarly journals Complex deletion and proximal reinsertion of a 150bp regulatory sequence in the mouse Csf1r promoter mediated by CRISPR-Cas9

2019 ◽  
Author(s):  
Kim M. Summers ◽  
Clare Pridans ◽  
Evi Wollscheid-Lengeling ◽  
Kathleen Grabert ◽  
Antony Adamson ◽  
...  
Keyword(s):  
Genome Editing ◽  
Copy Number ◽  
Large Scale ◽  
Regulatory Sequence ◽  
Genomic Deletion ◽  
Enhancer Region ◽  
Heterozygous Animal ◽  
A Genome ◽  
Pcr Products ◽  
Pcr Primer

AbstractThis paper describes a deletion/reinsertion event encountered in a genome-editing project using CRISPR-Cas9. The objective was to delete a 150bp enhancer region in the mouse Csf1r locus using a pair of guides and a homology-dependent repair (HDR) template. The editing was successful in generating a founder pup with the anticipated precise deletion. However, the deleted fragment and a duplicated copy of part of the HDR template was reinserted around 50bp downstream. The reinsertion event was recognised because the PCR primer site used in genotyping was duplicated, so that there were three PCR products in a heterozygous animal and two in a homozygote. The event we describe is more subtle and more difficult to detect than large-scale rearrangements reported by others. We suggest that any genomic deletion mediated by CRISPR-Cas9 needs to be confirmed by assessing the copy number in the genome.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Cell Line ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract Objective Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. Results We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

2018 ◽  
Author(s):  
Xu Tang ◽  
Guanqing Liu ◽  
Jianping Zhou ◽  
Qiurong Ren ◽  
Qi You ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Editing ◽  
Genome Sequencing ◽  
Large Scale ◽  
High Specificity ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Guide Rnas ◽  
A Genome ◽  
Multiple Sample

Targeting specificity has been an essential issue for applying genome editing systems in functional genomics, precise medicine and plant breeding. Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is critical for research and regulation. In plants, only limited studies had used whole-genome sequencing (WGS) to test off-target effects of Cas9. However, the cause of numerous discovered mutations is still controversial. Furthermore, WGS based off-target analysis of Cpf1 has not been reported in any higher organism to date. Here, we conducted a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice, a major food crop with a genome size of ~380 Mb. The sequencing depth ranged from 45X to 105X with reads mapping rate above 96%. Our results clearly show that most mutations in edited plants were created by tissue culture process, which caused ~102 to 148 single nucleotide variations (SNVs) and ~32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and 3 Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Taken together, our comprehensive and rigorous analysis of WGS big data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.

scholarly journals Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1828
Author(s):  
Francesco Citiulo ◽  
Cristina Crosatti ◽  
Luigi Cattivelli ◽  
Chiara Biselli
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Fast Response ◽  
Scale Production ◽  
Bacterial Cells ◽  
Regulatory Requirements ◽  
Emergency Situations ◽  
Large Scale Production ◽  
A Genome ◽  
Transformation Systems

The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles - VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.

scholarly journals Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

2018 ◽  
Author(s):  
Belen Gutierrez ◽  
Jérôme Wong Ng ◽  
Lun Cui ◽  
Christophe Becavin ◽  
David Bikard
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Mixed Population ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
A Genome

AbstractThe main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Cell Lines ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract ObjectiveGeneration of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any cell line.ResultsWe describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1604
Author(s):  
Jia Xin Tang ◽  
Angela Pyle ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Mitochondrial Diseases ◽  
Biological Research ◽  
Genetic Changes ◽  
Mitochondrial Proteome ◽  
A Genome ◽  
Indispensable Tool

Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.

0306 Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

2016 ◽  
Vol 94 (suppl_5) ◽  
pp. 146-146
Author(s):  
D. M. Bickhart ◽  
L. Xu ◽  
J. L. Hutchison ◽  
J. B. Cole ◽  
D. J. Null ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genetic Markers ◽  
Genome Sequencing ◽  
Copy Number ◽  
Large Scale ◽  
Copy Number Variants ◽  
Whole Genome

scholarly journals Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

2021 ◽  
Vol 22 (2) ◽  
pp. 480
Author(s):  
Jongrae Kim ◽  
Kwang Suk Chang ◽  
Sangmuk Lee ◽  
EonSeon Jin
Keyword(s):  
Genome Editing ◽  
Chlorella Vulgaris ◽  
Negative Selection ◽  
Screening Strategy ◽  
Feed Additive ◽  
Cell Factory ◽  
Dna Transformation ◽  
Research Groups ◽  
A Genome ◽  
Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

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