What is CRISPR/Cas9?

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CRISPR/Cas9 provides a precision tool for researchers to create SNPs, insertions, gene deletions and gene knockdowns in a wide array of experimental cell types. The emergence of this user-friendly technology represents a quantum leap forward in genome engineering.

The Role of Bacteria

First discovered by scientists studying the immune system of bacteria (specifically E. coli), CRISPR is based on the way the bacteria defends against virus infections. It does this by creating RNA that, when combined with a gene editing nuclease called Cas9, can target the DNA of an invading virus and disable it by cutting its gene sequence.

Researchers then later realized that this same method can be applied to the identification and editing of any DNA sequence, not just that of viruses, thereby opening up an entire world of genome engineering.

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Cas9

Known as the “molecular scissor”, Cas9 is a nuclease that is responsible for the cutting of a sequence of DNA that has been identified by a CRISPR guide RNA. The “cutting”, which occurs through a biochemical interaction, allows the gene to be disabled, repaired or altogether replaced with a new strand of DNA.

How CRISPR/Cas9 Works

CRISPR/Cas9 provides a precision tool for researchers to create SNPs, insertions, gene deletions and gene knockdowns in a wide array of experimental cell types. The emergence of this user-friendly technology represents a quantum leap forward in genome engineering.

A guide RNA, either as a crRNA:tracrRNA duplex or a sgRNA molecule, programs the Cas9 nuclease to cut at a specific genomic location. As is shown in the image, the programmable target-specific spacer portion of the sgRNA binds to the genomic target through RNA-DNA base pairing and initiates DNA strand cutting (denoted by scissors in the image). The target site in the genome must lie immediately 5′ of a PAM sequence that contains the canonical (5′) NGG (3′) specific for S. pyogenes Cas9.

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Cpf1 Alternative to Cas9

Cpf1 is a recently discovered Cas9 homolog that is smaller than Cas9 and utilizes a different PAM (Protospacer Adjacent Motif) to target different sequences. It is particularly suitable for AT-rich genomes/regions, and because it creates a staggered cut, it is useful for promoting HDR (Homology Directed Repair) when engineering SNPs and gene deletions.

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