Life Technologies

12 Life Technologies ™ | Genome editing Technology overview Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR- associated (Cas) proteins are derived from bacteria and archaea, where they are part of an adaptive immune system that protects the organism against invading DNA (Figure 2). Three-component CRISPR-Cas9 editing Genome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter the DNA in a cell. The CRISPR-Cas9 system takes advantage of a short guide RNA to target the bacterial Cas9 endonuclease to specific genomic loci. Because the guide RNA supplies the specificity, changing the target only requires a change in the design of the sequence that encodes the guide RNA. The CRISPR-Cas9 system used in gene editing consists of three components: the Cas nuclease Cas9 (a double-stranded DNA endonuclease), a target-complementary crRNA, and an auxiliary tracrRNA (see Figure 3). CRISPR-Cas9 technology Revolutionizing the field of genome editing Figure 2. CRISPR-Cas9 mechanism. Small segments of plasmids or viral genomes from an earlier infection are incorporated into CRISPR loci. CRISPR loci are transcribed and processed into short crRNAs that are complementary to previously encountered foreign DNA. The mechanism of target recognition relies on a sequence within the crRNA and a conserved sequence adjacent to the crRNA binding region called the protospacer-adjacent motif (PAM). A trans-activating crRNA (tracrRNA) pairs with a complementary pre-crRNA to form a duplex. That RNA duplex is cut by RNase III to form the so-called guide RNA (gRNA), a crRNA-tracrRNA chimera that guides Cas9 endonuclease to cleave and inactivate a new intruder.

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