Numerous publications address the skin sensitizing potential of the short chain alkanolamines triethanolamine (TEA), diethanolamine (DEA), monoethanolamine (MEA), which are not skin sensitizing ...according to animal studies. Regarding TEA, we analysed patch test data of 85 098 patients who had been tested with TEA 2.5% petrolatum by Information Network of Departments of Dermatology (IVDK) to identify particular exposures possibly associated with an elevated risk of sensitization. Altogether, 323 patients (0.4%) tested positive. The profile of patch test reactions indicates a slightly irritant potential rather than a true allergic response in many cases. Although used widely, no exposure associated with an increased risk of TEA sensitization was identified. Therefore, the risk of sensitization to TEA seems to be very low. MEA and DEA were patch tested in a much more aimed fashion in 9602 and 8791 patients, respectively when prevalence of contact allergy was 3.8% and 1.8%. MEA is the prominent allergen in metalworkers with exposure to water‐based metalworking fluids (wbMWFs); DEA is probably used in cutting fluids less frequently nowadays. Chronic damage to the skin barrier resulting from wbMWF, the alkalinity of ethanolamines (increasing from TEA to MEA), and other cofactors may contribute to a notable sensitization risk.
Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and ...even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.
The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we ...find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.
The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a ...CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.
Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle ...to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
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•CRISPR scaffold RNAs (scRNAs) encode both target locus and regulatory function•scRNAs function efficiently in mammalian and yeast cells•scRNAs enable transcription programs with simultaneous activation and repression•Combinatorial control of multiple genes enables flexible pathway manipulation
Modular CRISPR RNA scaffolds engineered to encode both guides to a target locus and recruitment of transcriptional regulators allow simultaneous gene activation and repression of multiple different genes in eukaryotic cells, greatly expanding the synthetic biology toolkit.
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To ...facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR–Cas9 guide design.
The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA ...guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.
Essential oils in perfumed products are important skin
sensitizers (1). Upon air exposure, lavender oil, with
the major components linalool and linalyl acetate, has
been shown to produce strongly ...allergenic hydroper-
oxides (2). Furthermore, linalool of natural origin was
found to contain a higher concentration of hydroperox-
ides than synthesized linalool (3). We have previously
studied autoxidation of sweet orange oil (main compound
R
-limonene)andpetitgrainoil(majorcompoundslinaloo and linalyl acetate) with liquid chromatography/mass
spectrometry/mass spectrometry (LC/MS/MS) (4). The
investigation was performed at room temperature, under
a daylight lamp (12hrs a day), and the oils were stirred
1hr four times a day, according to previous experience (5,
6). In the present study, the importance of handling con-
ditions for hydroperoxide formation was investigated. The
hydroperoxide concentrations of
R
-limonene, linalool
and linalyl acetate were analysed over time.
Cas12a is a bacterial RNA-guided nuclease used widely for genome editing and, more recently, as a molecular diagnostic. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, ...anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three such inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity via functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit recognition of double-stranded DNA (dsDNA), with AcrVA4 driving dimerization of Cas12a. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophages with tools to evade CRISPR-Cas12a and support biotechnological applications for which multiple-turnover enzymatic inhibition of Cas12a is desirable.
Summary
Microalgae are promising feedstock for biofuels yet mechanistic probing of their cellular network and industrial strain development have been hindered by lack of genome‐editing tools. ...Nannochloropsis spp. are emerging model microalgae for scalable oil production and carbon sequestration. Here we established a CRISPR/Cas9‐based precise genome‐editing approach for the industrial oleaginous microalga Nannochloropsis oceanica, using nitrate reductase (NR; g7988) as example. A new screening procedure that compares between restriction enzyme‐digested nested PCR (nPCR) products derived from enzyme‐digested and not‐digested genomic DNA of transformant pools was developed to quickly, yet reliably, detect genome‐engineered mutants. Deep sequencing of nPCR products directly amplified from pooled genomic DNA revealed over an 1% proportion of 5‐bp deletion mutants and a lower frequency of 12‐bp deletion mutants, with both types of editing precisely located at the targeted site. The isolated mutants, in which precise deletion of five bases caused a frameshift in NR translation, grow normally under NH4Cl but fail to grow under NaNO3, and thus represent a valuable chassis strain for transgenic‐strain development. This demonstration of CRISPR/Cas9‐based genome editing in industrial microalgae opens many doors for microalgae‐based biotechnological applications.
Significance Statement
Microalgae are promising feedstocks for biofuels, but additional genomic tools are needed to fully dissect the cellular networks underlying production of oil and biomass. Here, using nitrate reductase as an example, we demonstrate CRISPR/Cas9‐based genome editing for a model industrial microalga.
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