Decision letter: A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

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.

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A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

eLife ◽

10.7554/elife.55793 ◽

2020 ◽

Vol 9 ◽

Author(s):

Deepti Trivedi ◽

Vinitha CM ◽

Karishma Bisht ◽

Vishnu Janardan ◽

Awadhesh Pandit ◽

...

Keyword(s):

Human Disease ◽

Genome Engineering ◽

Cultured Cells ◽

Cellular Organization ◽

Cellular Processes ◽

Disease Biology ◽

A Genome ◽

Biochemical Analyses

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. 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.

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

10.21203/rs.2.21279/v1 ◽

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.

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Building a genome engineering toolbox in nonmodel prokaryotic microbes

Biotechnology and Bioengineering ◽

10.1002/bit.26727 ◽

2018 ◽

Vol 115 (9) ◽

pp. 2120-2138 ◽

Cited By ~ 16

Author(s):

Emily Freed ◽

Jacob Fenster ◽

Sharon L. Smolinski ◽

Julie Walker ◽

Calvin A. Henard ◽

...

Keyword(s):

Genome Engineering ◽

A Genome

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

Chemical Society Reviews ◽

10.1039/c6cs00197a ◽

2016 ◽

Vol 45 (24) ◽

pp. 6666-6684 ◽

Cited By ~ 16

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.

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DNA Editing: A Primer

Blood ◽

10.1182/blood.v126.23.sci-16.sci-16 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-16-SCI-16

Author(s):

Prashant Mali

Keyword(s):

Genome Engineering ◽

Conflicts Of Interest ◽

Cellular Organization ◽

Dna Editing ◽

Tools And Techniques ◽

And Behavior

Abstract RNA-guided Cas9 nucleases based on the prokaryotic CRISPR-Cas systems provide an unprecedented ease in being able to edit the genomes of diverse organisms. As a single unifying factor capable of co-localizing RNA, DNA, and protein, I believe tools and techniques based on Cas9 will grant exquisite control over cellular organization, regulation, and behavior. Here I will describe work on development of the CRISPR-Cas9 targeting methodology, and detail current and prospective genome-engineering methodologies. Disclosures No relevant conflicts of interest to declare.

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

Genes ◽

10.3390/genes10080596 ◽

2019 ◽

Vol 10 (8) ◽

pp. 596 ◽

Cited By ~ 6

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.

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

Microbial Cell Factories ◽

10.1186/s12934-019-1219-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 10

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.

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

Scientific Reports ◽

10.1038/s41598-021-82137-z ◽

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.

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