CRISPR technology overview
Clustered regulatory interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins are found in many bacteria and most archaea. The CRISPR-Cas systems use sequences derived from plasmids and phages to activate Cas endonucleases to neutralize those plasmids and phages via RNA-guided sequence-specific DNA cleavage, thus blocking their transmission and creating a simple acquired immunity.
With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell.
Three-component CRISPR editing
Genome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter the DNA in a cell. The CRISPR-Cas system takes advantage of a short guide RNA to target the bacterial Cas9 endonuclease to specific genomic loci. Because the specificity is supplied by the guide RNA, changing the target only requires a change in the design of the sequence encoding the guide RNA.
The CRISPR-Cas 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. The crRNA and tracrRNA of the GeneArt® CRISPR Nuclease Vector are expressed together as a guide RNA that mimics the natural crRNA-tracrRNA chimera in bacterial systems. You need only introduce a double-stranded oligonucleotide encoding a desired sequence that will serve to express the crRNA portion of the chimera.
CRISPR/Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 base pairs upstream of the NGG protospacer adjacent motif (PAM) sequence on the 3’ end of the target sequence.
Bench Tip Video: CRISPR/Cas Genome Editing Technology
In this Bench Tip Video, Dr. Mike Okimoto discusses CRISPR/Cas genome editing technology.CRISPR technology can be used to quickly and efficiently edit and manipulate a genomic locus in many different cell types and organisms. It can be used for a variety of genome modifications including gene deletion or knockout, knock-in, and knockdown. Watch this video to learn how to set up a CRISPR experiment and the applications of CRISPR technology.
If you haven’t heard of CRISPR, watch this recorded webinar to get an overview of CRISPR-Cas–mediated genome editing and learn about the GeneArt® CRISPR Nuclease System.