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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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A guide to genome engineering with programmable nucleases - Nature Rev. Gen.

A guide to genome engineering with programmable nucleases - Nature Rev. Gen. | molecular biology | Scoop.it

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Kim & Kim 2014

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.


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Fesquet didier's curator insight, May 28, 2014 8:59 AM

a review of these amazing technique

Rescooped by Jose Eduardo Ulloa Rojas from Plant immunity and legume symbiosis
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A Two-Hybrid-Receptor Assay Demonstrates Heteromer Formation as Switch-On for Plant Immune Receptors

A Two-Hybrid-Receptor Assay Demonstrates Heteromer Formation as Switch-On for Plant Immune Receptors | molecular biology | Scoop.it

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a “two-hybrid-receptor assay” is of more general use for studying heteromeric receptor complexes.

 

 


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Rescooped by Jose Eduardo Ulloa Rojas from Immunology
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ScienceDirect.com - Microbes and Infection - Cell biology of infection by Legionella pneumophila

ScienceDirect.com - Microbes and Infection - Cell biology of infection by Legionella pneumophila | molecular biology | Scoop.it
RT @MicrobesInfect: Microbes and Infection - Cell biology of infection by Legionella pneumophila (review article) http://t.co/rJyt8UBL

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Rescooped by Jose Eduardo Ulloa Rojas from Next Generation Sequencing (NGS)
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Sizing Up Gene Editing Tools

Sizing Up Gene Editing Tools | molecular biology | Scoop.it

Will a new approach for assessing genome-editing techniques increase the pace at which these technologies can be used for new applications? 


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Rescooped by Jose Eduardo Ulloa Rojas from Synthetic Biology
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Building 'smart' cell-based therapies

Building 'smart' cell-based therapies | molecular biology | Scoop.it

A technology for engineering human cells as therapies has been developed by scientists. The the technology becomes activated only in diseased tissues. It sits on the surface of a cell and can be programmed to sense specific external factors. For example, the engineered cell could detect big, soluble protein molecules that indicate that it's next to a tumor. When the biosensor detects such a factor, it sends a signal into the engineered cell's nucleus to activate a gene expression program, such as the production of tumor-killing proteins or chemicals.


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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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A TAL effector repeat architecture for frameshift binding : Nature Comm.

A TAL effector repeat architecture for frameshift binding : Nature Comm. | molecular biology | Scoop.it

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Richter et al, 2014

So far, TALE repeats are described to bind as a consecutive array to a consecutive DNA sequence, in which each repeat independently recognizes a single DNA base. This modular protein architecture enables the design of any desired DNA-binding specificity for biotechnology applications. Here we report that natural TALE repeats of unusual amino-acid sequence length break the strict one repeat-to-one base pair binding mode and introduce a local flexibility to TALE–DNA binding. This flexibility allows TALEs and TALE nucleases to recognize target sequence variants with single nucleotide deletions. The flexibility also allows TALEs to activate transcription at allelic promoters that otherwise confer resistance to the host plant.


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Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes - Curr. Opin. Biotech.

Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes - Curr. Opin. Biotech. | molecular biology | Scoop.it

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Copeland et al, 2014

TALEs, CRISPR/Cas, and sRNAs can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/Cas) or RNA (sRNAs) because of the simple rules governing their interactions with nucleic acids. This unique property enables these tools to repress the expression of genes at the transcriptional or post-transcriptional levels, respectively, without prior manipulation of cis-acting and/or chromosomal target DNA sequences. These tools are now being harnessed by synthetic biologists, particularly those in the eukaryotic community, for genome-wide regulation, editing, or epigenetic studies. Here we discuss the exciting opportunities for using TALEs, CRISPR/Cas, and sRNAs as synthetic trans-acting regulators in prokaryotes.


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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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Transcription Activator-like Effectors: A Toolkit for Synthetic Biology - ACS Synth. Biol.

Transcription Activator-like Effectors: A Toolkit for Synthetic Biology - ACS Synth. Biol. | molecular biology | Scoop.it

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Moore et al, 2014

Transcription activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria to aid the infection of plant species. TALEs assist infections by binding to specific DNA sequences and activating the expression of host genes. Recent results show that TALE proteins consist of a central repeat domain, which determines the DNA targeting specificity and can be rapidly synthesized de novo. Considering the highly modular nature of TALEs, their versatility, and the ease of constructing these proteins, this technology can have important implications for synthetic biology applications. Here, we review developments in the area with a particular focus on modifications for custom and controllable gene regulation.


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dromius's curator insight, March 13, 2014 4:30 AM

Table 2 gives an overview over assembly methods and their required time effort.

Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells - PLOS One

True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells - PLOS One | molecular biology | Scoop.it

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Gurushidze et al, 2014

In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.


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dromius's curator insight, March 19, 2014 11:01 AM

cool strategy leading to instanteously homozygous plants

Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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TALEN utilization in rice genome modifications

TALEN utilization in rice genome modifications | molecular biology | Scoop.it

Li et al, 2014

Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening.


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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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A guide to genome engineering with programmable nucleases - Nature Rev. Gen.

A guide to genome engineering with programmable nucleases - Nature Rev. Gen. | molecular biology | Scoop.it

(via T. Lahaye, thx)

Kim & Kim 2014

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.


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Fesquet didier's curator insight, May 28, 2014 8:59 AM

a review of these amazing technique

Rescooped by Jose Eduardo Ulloa Rojas from Next Generation Sequencing (NGS)
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University showcases next-generation sequencing research

University showcases next-generation sequencing research | molecular biology | Scoop.it

Symposium April 22 looks at deciphering DNA on a large scale


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Rescooped by Jose Eduardo Ulloa Rojas from Stem Cells, Regenerative Medicine & Tissue Engineering
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Cell Death and Differentiation - Immunobiology of mesenchymal stem cells

Cell Death and Differentiation - Immunobiology of mesenchymal stem cells | molecular biology | Scoop.it

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Jacob Blumenthal's curator insight, November 5, 2013 2:22 PM

Mesenchymal stem cells play an important role in cell therapy and are being currently tested in clinical trials for a variety of diseases. This open-access paper focus on their immunomodulatory effect.

 

Rescooped by Jose Eduardo Ulloa Rojas from Plant immunity and legume symbiosis
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Science: Paired Plant Immune Receptors (2014)

Science: Paired Plant Immune Receptors (2014) | molecular biology | Scoop.it

Plants are constantly interpreting microbial signals from potential pathogens and potential commensals or mutualists. Because plants have no circulating cells dedicated to this task, every plant cell must, in principle, recognize any microbe as friend, foe, or irrelevant bystander. That tall order is mediated by an array of innate immune system receptors: pattern-recognition receptors outside the plant cell and nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) inside the cell. Despite their importance for plant health, how NLRs function mechanistically has remained obscure. On page 299 of this issue, Williams et al. (1) reveal a role for heterodimerization between NLRs and show how the rather limited NLR repertoire of any plant genome might be enhanced by combinatorial diversity.

 

Marc T. Nishimura, Jeffery L. Dangl


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Rescooped by Jose Eduardo Ulloa Rojas from from Flow Cytometry to Cytomics
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Cell biology: Death by deacetylation - Nature.com

Cell biology: Death by deacetylationNature.comIt has long been thought that necrotic cell death is simply a passive process resulting from severe stress on a cell, due to infection or trauma, rather than a regulated mechanism.

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Rescooped by Jose Eduardo Ulloa Rojas from Genomics and metagenomics of microbes
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Highly Multiplexed Subcellular RNA Sequencing in Situ

Highly Multiplexed Subcellular RNA Sequencing in Situ | molecular biology | Scoop.it

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.


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Bradford Condon's curator insight, April 18, 2014 9:52 AM

WOW what a technique

Rescooped by Jose Eduardo Ulloa Rojas from DNA and RNA Research
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Identified epigenetic factors associated with an increased risk of developing cancer

Identified epigenetic factors associated with an increased risk of developing cancer | molecular biology | Scoop.it

In 10% of human tumors there is a family history of hereditary disease associated with mutations in identified genes. The best examples are the cases of polyps in the large intestine associated with the APC gene and breast cancer associated with BRCA1 and BRCA2 genes. In the remaining 90% of cases are believed to have an increased risk of developing cancer in relation to genetic variants less powerful but more often, for example, doubles the risk of having a tumor that lacks this small change, called polymorphisms.


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Rescooped by Jose Eduardo Ulloa Rojas from DNA and RNA Research
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Plant powerhouses are more than just energy producers

Plant powerhouses are more than just energy producers | molecular biology | Scoop.it

For more than a billion years, plants have had an internal dialogue, and we are just beginning to learn the words. The unusual dialogue occurs between two compartments within plant cells – the nucleus and the chloroplast. It is a dialogue that continues today, and, according to research just published in Science, it shapes the productivity of plants.


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Rescooped by Jose Eduardo Ulloa Rojas from Next Generation Sequencing (NGS)
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Stanford researchers create a new technique to study how we inherit disease

Stanford researchers create a new technique to study how we inherit disease | molecular biology | Scoop.it

By sequencing the DNA of the offspring, scientists can now determine which traits came from each parent during the gene-shuffling process of fertilization.


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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus - Biochem. BioPhys. Res. Comm.

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus - Biochem. BioPhys. Res. Comm. | molecular biology | Scoop.it

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Wu et al, 2014

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.


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Rescooped by Jose Eduardo Ulloa Rojas from TAL effector science
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A simple TALEN-based protocol for efficient genome-editing in Drosophila

A simple TALEN-based protocol for efficient genome-editing in Drosophila | molecular biology | Scoop.it

Zhang et al. 2014

Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centers. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately two months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.


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Top 25 Plasmids of All Time—Addgene Statistics Reveal Research Trends

Top 25 Plasmids of All Time—Addgene Statistics Reveal Research Trends | molecular biology | Scoop.it

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a list of the most requested plasmid in each year of Addgene’s history.  Over 800 plasmids in our collection have been distributed over 100 times.  There is amazing diversity in the fields of research covered in these 800 plasmids.  To get a sense of popular research technologies, let’s take a look at our top 25 requested plasmids of all time, listed in the order in which they became available through our service.


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dromius's curator insight, March 28, 2014 4:17 PM
" Our most popular kit of the year was the Golden Gate TALEN Kit, which continues to be our most requested kit of the past 10 years. "
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Targeted gene disruption in mammalian cell lines using programmable nucleases - AACR Education Book

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Kim 2014

Programmable nucleases, which include zinc finger nucleases (ZFNs) (1–4), transcription activator-like (TAL) effector nucleases (TALENs) (5, 6) and RNA-guided engineered nucleases (RGENs) (7–9) derived from the Type II CRISPR/Cas system, a prokaryotic immune response, are now widely used for gene knockout and knockin studies in cultured cells and model organisms. These nucleases cleave chromosomal DNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), whose repair via endogenous systems known as homologous recombination (HR) and non-homologous end joining (NHEJ) leads to targeted mutagenesis. Nuclease-mediated gene disruption is preferentially achieved via error-prone NHEJ rather than HR because NHEJ is a dominant DSB repair pathway over HR in mammalian cells. DSB repair by erroneous NHEJ is accompanied by small insertions and deletions (indels) at nuclease target sites, which can cause frameshift mutations in a protein-coding sequence. Unlike siRNA or shRNA that is limited by incomplete gene suppression, programmable nucleases enable complete gene disruption. Furthermore, these nucleases are much more specific than is siRNA or shRNA. Still, engineered nucleases can induce off-target mutations at sites that are highly homologous to on-target sites. Repair of off-target DNA cleavages in cells can cause gross chromosomal rearrangements such as deletions, inversions, and translocations (10, 11). In this session, I will compare these nucleases and discuss their pros and cons, focusing on their availability and off-target effects.


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