scholarly journals CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

2015 ◽  
Vol 1 (7) ◽  
pp. e1500454 ◽  
Author(s):  
Xin Han ◽  
Zongbin Liu ◽  
Myeong chan Jo ◽  
Kai Zhang ◽  
Ying Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Function Analysis ◽  
Embryonic Stem ◽  
Cell Types ◽  
Disease Genes ◽  
Deformation Method ◽  
Targeting Therapy ◽  
Membrane Deformation ◽  
Guide Rna ◽  
Different Cell Types

The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

scholarly journals MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1074
Author(s):  
Giuseppina Divisato ◽  
Silvia Piscitelli ◽  
Mariantonietta Elia ◽  
Emanuela Cascone ◽  
Silvia Parisi
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Epithelial Mesenchymal Transition ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cancer Development ◽  
Special Focus ◽  
Self Renewal ◽  
Mesenchymal Transition ◽  
Different Cell Types

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial–mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial–mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

scholarly journals Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanjie Luo ◽  
Ren Na ◽  
Julia S. Nowak ◽  
Yang Qiu ◽  
Qing Shi Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Delivery System ◽  
Gene Function ◽  
Function Analysis ◽  
Soybean Plant ◽  
Apple Latent Spherical Virus ◽  
Guide Rna ◽  
Guide Rnas ◽  
Plant Trait ◽  
Trait Improvement

Abstract Background A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. Results To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. Conclusions With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

scholarly journals Nuclear Receptors in Regulation of Mouse ES Cell Pluripotency and Differentiation

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-10 ◽  
Author(s):  
Eimear M. Mullen ◽  
Peili Gu ◽  
Austin J. Cooney
Keyword(s):  
Nuclear Receptors ◽  
Therapeutic Potential ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cell Phenotype ◽  
Es Cell ◽  
Complex Signaling ◽  
Different Cell Types ◽  
Mouse Es Cell

Embryonic stem (ES) cells have great therapeutic potential because they are capable of indefinite self-renewal and have the potential to differentiate into over 200 different cell types that compose the human body. The switch from the pluripotent phenotype to a differentiated cell involves many complex signaling pathways including those involving LIF/Stat3 and the transcription factors Sox2, Nanog and Oct-4. Many nuclear receptors play an important role in the maintenance of pluripotence (ERRβ, SF-1, LRH-1, DAX-1) repression of the ES cell phenotype (RAR, RXR, GCNF) and also the differentiation of ES cells (PPARγ). Here we review the roles of the nuclear receptors involved in regulating these important processes in ES cells.

scholarly journals Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Sergey Rodin ◽  
Liselotte Antonsson ◽  
Colin Niaudet ◽  
Oscar E. Simonson ◽  
Elina Salmela ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
A Cell ◽  
Hes Cells ◽  
Free Environment ◽  
Different Cell Types ◽  
E Cadherin

Abstract Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

scholarly journals Human telomeres replicate using chromosome-specific, rather than universal, replication programs

2012 ◽  
Vol 197 (2) ◽  
pp. 253-266 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Zi Yan ◽  
Simone G. Calderano ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Embryonic Stem ◽  
Cell Types ◽  
Es Cell ◽  
Site Location ◽  
Subtelomeric Heterochromatin ◽  
Basic Features ◽  
Different Cell Types ◽  
Single Molecule Analysis ◽  
Individual Human

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs—i.e., origin distribution, termination site location, and fork rate and direction—of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

scholarly journals CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

2019 ◽  
Vol 47 (W1) ◽  
pp. W171-W174 ◽  
Author(s):  
Kornel Labun ◽  
Tessa G Montague ◽  
Maximilian Krause ◽  
Yamila N Torres Cleuren ◽  
Håkon Tjeldnes ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Alternative Transcript ◽  
Command Line ◽  
Web Tool ◽  
Guide Rna ◽  
Dna Targeting ◽  
Long Read ◽  
Targeted Enrichment ◽  
Crispr Activation

Abstract The CRISPR–Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR–Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR–Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no

scholarly journals Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

2020 ◽  
Vol 21 (19) ◽  
pp. 7353 ◽  
Author(s):  
Karim Shalaby ◽  
Mustapha Aouida ◽  
Omar El-Agnaf
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Viral Vectors ◽  
Scale Up ◽  
Cell Types ◽  
Delivery Systems ◽  
Different Cell Types ◽  
Immunogenic Response ◽  
Cas Gene

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.

scholarly journals Rapid interrogation of cancer cell of origin through CRISPR editing

2021 ◽  
Vol 118 (32) ◽  
pp. e2110344118
Author(s):  
Weiran Feng ◽  
Zhen Cao ◽  
Pei Xin Lim ◽  
Huiyong Zhao ◽  
Hanzhi Luo ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Ex Vivo ◽  
Cell Types ◽  
Genetically Engineered ◽  
Cell Of Origin ◽  
Guide Rna ◽  
Immunodeficient Mice ◽  
Complex Technology ◽  
Different Cell Types

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9–sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

scholarly journals Comprehensive analysis of prime editing outcomes in human embryonic stem cells

2021 ◽  
Author(s):  
Omer Habib ◽  
Gizem Habib ◽  
Gue-ho Hwang ◽  
Sangsu Bae
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Cytidine Deaminase ◽  
Embryonic Stem ◽  
Cell Types ◽  
Great Promise ◽  
Nucleotide Substitutions ◽  
Base Editing

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

scholarly journals Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

2016 ◽  
Author(s):  
Christopher D Richardson ◽  
Grahm J Ray ◽  
Jacob E Corn
Keyword(s):  
Cell Lines ◽  
Gene Disruption ◽  
Genomic Sequence ◽  
Cell Types ◽  
Polyploid Cell ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Specific Manner ◽  
A Cell ◽  
Different Cell Types

Cas9 endonuclease can be targeted to genomic sequences by varying the sequence of the single guide RNA (sgRNA). The activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines, or using inefficient sgRNAs, can require extensive downstream screening to identify homozygous clones. We have found that linear, non-homologous oligonucleotide DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation greatly increases the frequency of clones with homozygous gene disruptions, even in polyploid cell lines, and rescues otherwise ineffective sgRNAs. The mechanism of enhanced gene disruption differs between human cell lines, stimulating deletion of genomic sequence and/or insertion of non-homologous oligonucleotide DNA at the edited locus in a cell line specific manner. Thus, the addition of non-homologous DNA appears to drive cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

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