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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.
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.
CRISPR [43] is the leading genetic engineering method. [44] In 2014, Esvelt and coworkers first suggested that CRISPR/Cas9 might be used to build gene drives. [ 5 ] In 2015, researchers reported successful engineering of CRISPR-based gene drives in Saccharomyces [ 45 ] , Drosophila , [ 46 ] and mosquitoes .
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Ishino was born in Kyoto Prefecture, Japan.He received his BS, MS and PhD in 1981, 1983 and 1986, respectively, from Osaka University. [1] From 1987 to 1989, he served as a post-doctoral fellow in Dieter Söll's laboratory at Yale University.
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]