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CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. [1] It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim , Adam Arkin, Jonathan Weissman , and Jennifer Doudna . [ 2 ]
CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [10] was the AAAS's choice for Breakthrough of the Year in 2015. [11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [12]
RT-LAMP does not require thermal cycles (unlike PCR) and is performed at a constant temperature between 60 and 65 °C. RT-LAMP is used in the detection of RNA viruses (groups II, IV, and V on the Baltimore Virus Classification system), such as the SARS-CoV-2 virus [ 4 ] and the Ebola virus .
The first stage involves the extension of bases in the CRISPR locus region by addition of foreign DNA spacers in the genome sequence. Proteins like cas1 and cas2, assist in finding new spacers. The next stage involves transcription of CRISPR: pre-crRNA (precursor CRISPR RNA) are expressed by the transcription of CRISPR repeat-spacer array.
See: Guide RNA, CRISPR. Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9. A small guide RNA (sgRNA), or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets. gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions.
CRISPR RNA or crRNA is a RNA transcript from the CRISPR locus. [1] CRISPR-Cas (clustered, regularly interspaced short palindromic repeats - CRISPR associated systems) is an adaptive immune system found in bacteria and archaea to protect against mobile genetic elements , like viruses , plasmids , and transposons . [ 2 ]
There are two main requirements for mRNA degradation to take place: a near-perfect complementary match between the guide strand and target mRNA sequence, and, a catalytically active Argonaute protein, called a 'slicer', to cleave the target mRNA. [1] There are two major pathways of mRNA degradation once cleavage has occurred.
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and open up 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 living organisms.