scholarly journals Building a genome engineering toolbox in nonmodel prokaryotic microbes

2018 ◽  
Vol 115 (9) ◽  
pp. 2120-2138 ◽  
Author(s):  
Emily Freed ◽  
Jacob Fenster ◽  
Sharon L. Smolinski ◽  
Julie Walker ◽  
Calvin A. Henard ◽  
...  
Keyword(s):  
Genome Engineering ◽  
A Genome

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

2016 ◽  
Vol 45 (24) ◽  
pp. 6666-6684 ◽  
Author(s):  
Amrita Singh ◽  
Debojyoti Chakraborty ◽  
Souvik Maiti
Keyword(s):  
Genome Editing ◽  
Chemical Biology ◽  
Genome Engineering ◽  
A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

scholarly journals A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling

2020 ◽  
Author(s):  
Deepti Trivedi ◽  
Vinitha CM ◽  
Karishma Bisht ◽  
Vishnu Janardan ◽  
Awadhesh Pandit ◽  
...  
Keyword(s):  
Human Disease ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Cellular Organization ◽  
Cellular Processes ◽  
Lipid Signalling ◽  
Disease Biology ◽  
A Genome ◽  
Biochemical Analyses

SummaryPhosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

scholarly journals Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 596 ◽  
Author(s):  
Haroon Butt ◽  
Agnieszka Piatek ◽  
Lixin Li ◽  
Anireddy S. N. Reddy ◽  
Magdy M. Mahfouz
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Genome Engineering ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Plant Genome ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
A Genome

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.

scholarly journals Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wei Shen ◽  
Jun Zhang ◽  
Binan Geng ◽  
Mengyue Qiu ◽  
Mimi Hu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Strain Improvement ◽  
Genomic Research ◽  
Francisella Novicida ◽  
A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

scholarly journals Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Navarro-Guerrero ◽  
Chwen Tay ◽  
Justin P. Whalley ◽  
Sally A. Cowley ◽  
Ben Davies ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Genome Engineering ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Loss Of Function ◽  
Lentiviral Transduction ◽  
Genome Wide ◽  
A Genome ◽  
Human Pathology ◽  
Induced Pluripotent

AbstractGenome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes—HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.

scholarly journals Functional analysis of caspase cleavable proteoforms from the Drosophila GSK-3 gene shaggy

2020 ◽  
Author(s):  
Dagmara Korona ◽  
Daniel Nightingale ◽  
Bertrand Fabre ◽  
Michael Nelson ◽  
Bettina Fischer ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Genome Engineering ◽  
Glycogen Synthase ◽  
Kinase Domain ◽  
Protein Isoforms ◽  
Cell Junction ◽  
Loss Of Function ◽  
Caspase Cleavage ◽  
Terminal Domain ◽  
A Genome

AbstractThe Drosophila shaggy (sgg) gene encodes the major fly orthologue of Glycogen Synthase Kinase −3 (GSK-3), a key highly conserved kinase at the heart of many signalling pathways. The sgg locus is complex, encoding multiple protein isoforms that are expressed in distinct temporal and tissue-specific patterns across development. Its isoforms predominantly differ at the carboxy and amino termini due to the use of different transcriptional start sites and alternative splicing events that include internal and terminal exons. One interesting class of proteins isoforms is represented by the Sgg-PD class (Sgg46), three proteoforms that contain a large 582 amino acid N-terminal domain which contains recognition sites for caspase-mediated cleavage. Regulated cleavage at these sites by non-apoptotic caspases has previously been implicated in the regulation of Sgg activity in adult bristle development. Here, we take a genome engineering approach to introduce specific tags into this unique Sgg-PD exon and utilise these for localisation and protein interaction studies. We also generated new loss of function alleles and specific mutations in the caspase cleavage motifs. We find that loss of functions Sgg-PD class alleles are viable and fertile, but exhibit adult locomotor and bristle defects. Expression analysis of lines carrying tags on both sides of the caspase cleavage sites indicates that the cleavage is developmentally regulated during embryogenesis. Surprisingly, we found that in some cells, particularly embryonic hemocytes, the N-terminal domain released by caspase cleavage is retained while the polypeptide containing the conserved kinase domain is apparently lost. Transcriptomic analysis of embryos homozygous for the new caspase-insensitive allele indicates a role for Sgg-PD in the regulation of cytoskeletal and cell junction functions, which is supported by proteomics analysis using specific in locus tags to identify common and unique protein interaction partners with N- and C-terminal domains. Taken together, our work identifies new activities for the Sgg protein and uncovers unexpected roles for caspase cleavage in Sgg biology.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background: The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.Results: We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.Conclusions: This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

2020 ◽  
Author(s):  
Xander Nuttle ◽  
Nicholas D. Burt ◽  
Benjamin Currall ◽  
Mariana Moysés-Oliveira ◽  
Kiana Mohajeri ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Single Cells ◽  
Functional Characterization ◽  
Copy Number Variants ◽  
Single Copy ◽  
Segmental Duplications ◽  
Individual Gene ◽  
Genomic Deletions ◽  
A Genome ◽  
Human Ipscs

Novel gene and variant discoveries have reached unprecedented scale with the emergence of exome and genome sequencing studies across a spectrum of human disease initiatives. Highly parallelized functional characterization of these variants is now paramount to deciphering disease mechanisms, and approaches that facilitate editing of induced pluripotent stem cells (iPSCs) to derive otherwise inaccessible tissues of interest (e.g., brain) have become critical in genomics research. Here, we sought to facilitate scalable editing of multiple genes and variants by developing a genome engineering approach that incorporates libraries of CRISPR/Cas9 guide RNAs (gRNAs) into a piggyBac (PB) transposon system. To test the efficiency of inducing small indels, targeted deletions, and large reciprocal copy number variants (CNVs), we simultaneously delivered to human iPSCs both Cas9 and a library including 59 single gRNAs targeting segmental duplications, 70 paired gRNAs flanking particular genic regions, and three single gRNAs targeting the coding sequence of an individual gene, MAGEL2. After editing, we isolated single cells, expanded resultant colonies, and genotyped their gRNA contents and mutational outcomes. We observed that 97.7% of gRNA constructs were integrated into at least one colony, with 85.6% of colonies containing three or fewer PB integrations. This PB editing method generated 354 cell lines with 57.8% of sequenced gRNA cleavage sites modified in at least one line, 14.4% of these lines altered at multiple targets, and single-copy indel mutagenesis predominating. Among the edits generated were eight targeted genomic deletions, including pathogenic microdeletions at chromosome 15q11-q13 (∼5.3 Mbp), chromosome 16p11.2 (∼740 kbp), and chromosome 17q11.2 (∼1.4 Mbp). These data highlight PB editing as a powerful platform for gene inactivation and testify to its strong potential for oligogenic modeling. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of near-isogenic cellular collections and catalyze comparative functional genomic studies, better positioning us to investigate the roles of hundreds of genes and mutations in development and disease.

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