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CRISPR gene editing (CRISPR, pronounced / ˈ k r ɪ s p ə r / (crisper), refers to a clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified.
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.
Targeted gene knockout using CRISPR/Cas9 requires the use of a delivery system to introduce the sgRNA and Cas9 into the cell. Although a number of different delivery systems are potentially available for CRISPR, [37] [38] genome-wide loss-of-function screens are predominantly carried out using third generation lentiviral vectors.
For example, the CRISPR-seq paper demonstrated the feasibility of in vivo studies using this technology, and the CROP-seq protocol facilitates large screens by providing a vector that makes the guide RNA itself readable (rather than relying on expressed barcodes), which allows for single-step guide RNA cloning. [6]
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases) system was originally discovered to be an acquired immune response mechanism used by archaea and bacteria. It has since been adopted for use as a tool in the genetic engineering of higher organisms.
The CRISPR/Cas system allows bacteria and archaea to fight against invading viruses by cleaving viral DNA and inserting pieces of that DNA into their own genome. The organism then transcribes this DNA into RNA and combines this RNA with Cas9 proteins to make double-stranded breaks in the invading viral DNA.
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 ]
Gene knock-in originated as a slight modification of the original knockout technique developed by Martin Evans, Oliver Smithies, and Mario Capecchi.Traditionally, knock-in techniques have relied on homologous recombination to drive targeted gene replacement, although other methods using a transposon-mediated system to insert the target gene have been developed. [3]
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