CRISPR (/ˈkrɪspər/) (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes.
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
Identification
CRISPRs are widely distributed among bacteria and archaea and show some sequence similarities. Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match.
Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content. However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase chain reaction (PCR) primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples; amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays.
The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely de novo identification or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) and direct repeat sequences from published genomes as a hook for identifying direct repeats in individual reads.
CRISPR Gene Editing
CRISPR technology has been applied in the food and farming industries to engineer probiotic cultures and to immunize industrial cultures (for yogurt, for instance) versus infections. It is also being used in crops to enhance yield, drought tolerance and nutritional homes.
By the end of 2014 some 1000 research papers had been published that mentioned CRISPR. The technology had been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains. CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria. CRISPR based approaches utilizing Cas12a have recently been utilized in the successful modification of a broad number of plant species.
In July 2019, doctors in Tennessee, United States, used CRISPR to experimentally treat a patient with a genetic disorder. The patient was a 34-year-old woman with sickle cell disease.
In the future, CRISPR gene editing could be used to create new species or revive extinct species from closely related ones.
CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies.
CRISPR as a Diagnostic Tool
CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences. In 2016, the Cas9 nuclease was used to deplete unwanted nucleotide sequences in next-generation sequencing libraries while requiring only 250 pg of initial RNA input. Beginning in 2017, CRISPR
associated nucleases were also used for direct diagnostic testing, down to single molecule sensitivity.
In 2019, some new diagnostic applications using CRISPR technology, an electrical CRISPR-Chip for the detection of genetic mutations and a microfluidic CRISPR-Biosensor for electrochemical miRNA diagnostics, were introduced.
https://en.wikipedia.org/wiki/CRISPR
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