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CRISPR Genome Editing


CRISPR Cas9 gene editing tools have been adapted from a prokaryotic immune system to provide sequence specfic genome targeting of any species.  The nuclease introduces double stranded breaks.  The break is repaired by one of two mechanisms: 1. Non-homologous end joining (NHEJ) which creates random insertions or deletions (indels) at the targetd site or 2. Homologous recombination which creates precise changes based on template DNA.    

CRISPR Cas9 for Genome Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems were originally discovered as part of a prokaryotic adaptive immune system to protect against invading viruses and bacteriophages.  The type II CRISPR/Cas system found in Streptococcus pyogenes has been well-studied, and is comprised of a CRISPR-associated (Cas9) endonuclease that complexes with two small guide RNAs, crRNA and tracrRNA, to make a double-stranded DNA break (DSB) in a sequence specific manner (Reviewed in Charpentier & Doudna, 2013).   The crRNA and tracrRNA, which can be combined into a single guide RNA (gRNA), directs the Cas9 nuclease to the target sequence through base pairing between the gRNA sequence and the genomic target sequence.  The target sequence consists of a 20-bp DNA sequence complementary to the gRNA, followed by trinucleotide sequence (5'-NGG-3') called the protospacer adjacent motif (PAM). The Cas9 nuclease digests both strands of the genomic DNA 3-4 nucleotides 5' of the PAM sequence.  By simply introducing different guide RNA sequences, the Cas9 can be programmed to introduce site-specific DNA double-strand breaks virtually anywhere in the genome where a PAM sequence is located.  The double-stranded break at the target site induces DNA repair mechanisms, such as non-homologous end joining (NHEJ) that create insertions and deletion (indels) leading to a premature stop codon, and homology-directed recombination (HR) for introducing or knocking in new sequences (Figure 1).  
  1. Non-homologous end joining (NHEJ) - In NHEJ, ends of the DNA are directly ligated back together.  The error prone process often inserts or deletes (InDels) nucleotides at the point of repair.  The InDels can cause frameshifts in the transcript leading to truncation and loss of protein function.
  2. Homologous recombination (HR) - In HR, a DNA template is introduced to the cell with sequences that are homologous to the areas flanking the target site.  During recombination, the homologous sequences are inserted into the target area with any intervening sequences between them.  This allows the introduction of precise mutations to the area. Often, the mutation includes the insertion of a selection marker and disrupts the gene resulting in a knockout allele. 

Paired gRNA strategies for added specificity
While the Cas9 system is a efficient and flexible system for genome editing, there is the potential for gRNA to target more than the intended site resulting in off-target effects (Ran et al. 2013). In order to compensate for this, the Cas9 nuclease was altered by the introduction of a D10A mutation.   The resulting nuclease introduces a single strand break, or 'nick,' to the DNA.  The Cas9(D10A) nickase requires two target sites in close proximity to induce the repair mechanisms.  However, the likelihood of off-targets is decreased significantly (Ran et al. 2013).

 

Find and Purchase CRISPR-Cas9

transEDIT CRISPR-Cas9 reagents targeting human, mouse and rat genomes.
Available targeting individual genes, gene families and pathways or the genome.

 
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