CRISPR/Cas in genome defense and gene editing

AbstractTargeted genome editing using engineered nucleases such as ZFNs and TALENs has been rapidly replaced by the CRISPR/Cas9 (clustered, regulatory interspaced, short palindromic/ CRISPR-associated nuclease) system. CRISPR/Cas9 technology represents a significant improvement enabling a new level of targeting, efficiency and simplicity. Gene editing mediated by CRISPR/Cas9 has been recently used not only in bacteria but in many eukaryotic cells and organisms, from yeasts to mammals. Other modifications of the CRISPR-Cas9 system have been used to introduce heterologous domains to regulate gene expressions or label specific loci in various cell types. The review focuses not only on native CRISPR/Cas systems which evolved in prokaryotes as an endogenous adaptive defense mechanism against foreign DNA attacks, but also on the CRISPR/Cas9 adoption as a powerful tool for site-specific gene modifications in fungi, plants and mammals.

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Advances in site-specific gene editing for primary immune deficiencies

Current Opinion in Allergy and Clinical Immunology ◽

10.1097/aci.0000000000000483 ◽

2018 ◽

Vol 18 (6) ◽

pp. 453-458 ◽

Cited By ~ 3

Author(s):

Caroline Y. Kuo

Keyword(s):

Gene Editing ◽

Specific Gene ◽

Site Specific ◽

Immune Deficiencies

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Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

Science Advances ◽

10.1126/sciadv.aay6687 ◽

2020 ◽

Vol 6 (12) ◽

pp. eaay6687 ◽

Cited By ~ 4

Author(s):

Haojie Sun ◽

Su Fu ◽

Shuang Cui ◽

Xiangsha Yin ◽

Xiaoyan Sun ◽

...

Keyword(s):

Rat Brain ◽

Gene Editing ◽

High Efficiency ◽

Protein A ◽

Cell Types ◽

Cell Labeling ◽

Specific Gene ◽

Creb Binding Protein ◽

A Genome ◽

And Function

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)–associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system–based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.

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Effect of local anesthetics on viability and differentiation of various adult stem/progenitor cells

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01905-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Young Hoon Kim ◽

Ga Young Park ◽

Nechama Rabinovitch ◽

Solaiman Tarafder ◽

Chang H. Lee

Keyword(s):

Progenitor Cells ◽

Local Anesthetics ◽

Adipogenic Differentiation ◽

Cell Types ◽

Dose Effect ◽

Specific Gene ◽

Gene Expressions ◽

Differentiation Capacity ◽

Matrix Deposition ◽

Lineage Differentiation

Abstract Background Local anesthetics (LAs) are widely used to control pain during various clinical treatments. One of the side effects of LAs, cytotoxicity, has been investigated in various cells including stem/progenitor cells. However, our understanding of the effects of LAs on the differentiation capacity of stem/progenitor cells still remains limited. Therefore, a comparative study was conducted to investigate the effects of multiple LAs on viability and multi-lineage differentiation of stem/progenitor cells that originated from various adult tissues. Method Multiple types of stem/progenitor cells, including bone marrow mesenchymal stem/progenitor cells (MSCs), dental pulp stem/progenitor cells (DPSCs), periodontal ligament stem/progenitor cells (PDLSCs), and tendon-derived stem/progenitor cells, were either obtained from a commercial provider or isolated from adult human donors. Lidocaine (LD) and bupivacaine (BP) at various doses (1×, 0.75×, 0.5×, and 0.25× of each physiological dose) were applied to the different stem/progenitor cells for an hour, followed by induction of fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation. Live/dead and MTT assays were performed at 24 h after the LD or BP treatment. At 2 weeks, qRT-PCR was conducted to evaluate the gene expressions associated with differentiation. After 4 weeks, multiple biochemical staining was performed to evaluate matrix deposition. Results At 24 h after LD or BP treatment, 1× and 0.75× physiological doses of LD and BP showed significant cytotoxicity in all the tested adult stem/progenitor cells. At 0.5×, BP resulted in higher viability than the same dose LD, with variance between cell types. Overall, the gene expressions associated with fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation were attenuated in LD or BP pre-treated stem/progenitor cells, with notable dose-effect and dependence on types. In contrast, certain doses of LD and/or BP were found to increase specific gene expression, depending on the cell types. Conclusion Our data suggest that LAs such as LD and BP affect not only the viability but also the differentiation capacity of adult stem/progenitor cells from various anatomical sites. This study sheds light on stem cell applications for tissue regeneration in which isolation and transplantation of stem cells frequently involve LA administration.

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A positive readout single transcript reporter for site-specific mRNA cleavage

PeerJ ◽

10.7717/peerj.3602 ◽

2017 ◽

Vol 5 ◽

pp. e3602

Author(s):

Nikolay Kandul ◽

Ming Guo ◽

Bruce A. Hay

Keyword(s):

Regulation Of Gene Expression ◽

Eukaryotic Cells ◽

Rna Cleavage ◽

Site Specific ◽

Coding Sequence ◽

Mrna Cleavage ◽

Genome Defense ◽

Translational Activation ◽

Single Transcript ◽

Specific Mrna

Cleavage of mRNA molecules causes their rapid degradation, thereby playing an important role in regulation of gene expression and host genome defense from viruses and transposons in bacterial and eukaryotic cells. Current negative-readout, and repressor-based positive-readout reporters of mRNA degradation have limitations. Here we report the development of a single transcript that acts as a positive reporter of mRNA cleavage. We show that placement of bacterial CopT and CopA hairpins into the 5′ UTR and 3′ UTR of an mRNA results in inhibition of translation of the intervening coding sequence inDrosophila. An internal poly(A) tract inserted downstream of the coding sequence stabilizes transcripts cut within the 3′ UTR. When these components are combined in a transcript in which targets sites for RNA cleavage are placed between the poly(A) tract and CopA, cleavage results in translational activation, providing a single transcript-based method of sensing mRNA cleavage with a positive readout.

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