Detection of CRISPR adaptation

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

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Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Nature Communications ◽

10.1038/s41467-019-12417-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 11

Author(s):

Anna A. Shiriaeva ◽

Ekaterina Savitskaya ◽

Kirill A. Datsenko ◽

Irina O. Vvedenskaya ◽

Iana Fedorova ◽

...

Keyword(s):

Escherichia Coli ◽

Nucleic Acid ◽

High Throughput Sequencing ◽

Dna Degradation ◽

Type I ◽

Dna Fragments ◽

Crispr Array ◽

Foreign Dna ◽

And Function

Abstract Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

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Tagging and Capturing of Lentiviral Vectors Using Short RNAs

International Journal of Molecular Sciences ◽

10.3390/ijms221910263 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10263

Author(s):

Martin Panigaj ◽

Michael P. Marino ◽

Jakob Reiser

Keyword(s):

Nucleic Acid ◽

Rna Binding ◽

Binding Domain ◽

Rna Aptamer ◽

Rna Sequences ◽

Rna Binding Domain ◽

Transgene Delivery ◽

Vector Particles ◽

Nucleic Acid Sequences

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

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Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA

Journal of Bioenergetics and Biomembranes ◽

10.1007/s10863-014-9543-2 ◽

2014 ◽

Vol 46 (2) ◽

pp. 147-156 ◽

Cited By ~ 6

Author(s):

Jaroslav Zelenka ◽

Lukáš Alán ◽

Martin Jabůrek ◽

Petr Ježek

Keyword(s):

Mitochondrial Dna ◽

Nucleic Acid ◽

5S Rrna ◽

Dna And Rna ◽

Nucleic Acid Sequences

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Gold-Aptamer-Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

10.26434/chemrxiv.8312324.v1 ◽

2019 ◽

Author(s):

Veeren Chauhan ◽

Mohamed M Elsutohy ◽

C Patrick McClure ◽

Will Irving ◽

Neil Roddis ◽

...

Keyword(s):

Nucleic Acid ◽

In Silico ◽

Point Of Care ◽

Lateral Flow ◽

Nucleic Acid Sequence ◽

In Silico Screening ◽

Lateral Flow Assays ◽

Life Threatening ◽

Software And Hardware ◽

Nucleic Acid Sequences

Enteroviruses are a ubiquitous mammalian pathogen that can produce mild to life-threatening disease. Bearing this in mind, we have developed a rapid, accurate and economical point-of-care biosensor that can detect a nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and aptamers to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral RNA sequence (23 bases), which was identified through in silico screening. Aptamers were designed to demonstrate specific complementarity towards the target enteroviral RNA to produce aggregated gold-aptamer nanoconstructs. Conserved target enteroviral nucleic acid sequence (≥ 1x10-7 M, ≥1.4×10-14 g/mL), initiates gold-aptamer-nanoconstructs disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow-assays that utilise gold-aptamer-nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (< 60 s) and could be interpreted with a bespoke software and hardware electronic interface. We anticipate our methodology will translate in-silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance.

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