TRANSFECTION TIPS VIDEO: CRISPR/Cas9 Transfection
This video covers the basic mechanism of CRISPR/Cas9 genome editing and introduces the key points affecting researchers' decisions about whether to deliver Cas9 and guide RNA in plasmid, RNA or RNP format.
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Overview of Genome Editing with CRISPR/Cas9
BACKGROUND
Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-stranded breaks in target DNA. Target cleavage by Cas9 requires base-pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA (See figure CRISPR/Cas9 Genome Editing). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.
EDITING MAMMALIAN GENOMES WITH CRISPR
The bacterial CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA. Once the Cas9 has cleaved the target DNA, two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), are triggered in response to the DNA break. The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. HDR requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence flanked on either side by sequences bearing homology to the target.
ADVANTAGES
The simplicity of using of a noncoding RNA guide to target DNA for site-specific cleavage provides a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Using the CRISPR/Cas9 strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of DNA-binding proteins.