CRISPR-Cas9

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I. Introduction

CRISPR-Cas 9 was discovered by investigators studying the immune response of bacteria to bacteriophages (viruses that infect bacteria).  Now its studied and used predominantly as potentially revolutionary and inexpensive method to genetically edit and DNA site imaginable

 

For supporting information about the CRISPR-Cas 9 gene editing complex, visit Biochemistry Online: Chapter 7B: Mechanisms of Enzyme Catalysis - CRISPR

General Structure

For supporting information about the CRISPR-Cas 9 gene editing complex, visit Biochemistry Online: Chapter 7B: Mechanisms of Enzyme Catalysis - CRISPR

This CRISP-Cas 9 complex consists of a nuclease called Cas 9 ( CRISPR associated protein), a single guide RNA (sg RNA) that determines specificity and single stranded target DNA. In bacteria, the RNA is an actual complex of two noncoding and interacting RNAs, crRNA and trans-activating CRISPR RNA tracRNA.  The sg-RNA is a covalently fused version of the crRA and trac RNA.

The Cas 9 apoenzyme

Cas 9 is an endonuclease that cleaves both strands of DNA 3 base pairs from a DNA motif, NCC/NGG called PAM ( protospacer adjacent motif) .  It has two distinct lobes. One is the nuclease lobe (NUC), amino acids 1-56 and 718-1368, which has two different nuclease domains for the two cleavages. The recognition or receptor lobe (REC), amino acids 94-717, interacts with the RNA molecules.  There is also a Arg-rich bridge helix (57-93)

lobes: NUC (nuclease, orange);  REC (receptor, recognition, light blue); Arg-rich bridge helix, red

The HNH-like nuclease domain cleaves the "target" DNA strand which is complementary to the crispr-RNA that confers specificity to the enzyme; the key catalytic residues are His 840 and Asn 854. It also contains a Mg ion and possible a second one as well.

The Ruv-like domain cleaves the complementary "non-target" strand with key active site residues Asp 10, Glu 762, Asp 986 and His 983.  It also contains a bound Mn ion.

HNH and Ruv active site residues: NUC (nuclease, orange);  REC (receptor, recognition, light blue); Arg-rich bridge helix, red

The Cas 9:sgRNA:target DNA ternary complex

The structure below shows the holo-Cas 9 complex with a single guide RNA (the tracRNA and crRNA from the CRISPR operon combined into 1 RNA molecule) and the 20 nucleotide host target DNA strand (not including the nontarget complementary host strand).  The structures shown above are also shown in the holo structure. 

sg RNA (gold); single stranded target DNA (green); PAM recognition site in protein (Arg 1333, 1335, spacefill).  Note:  the target DNA strand does not contain a PAM site in this crystal structure.

The structure below show just the nucleic acids and key amino acids (spacefill) from the protein involved in catalysis and binding.  Notice the heteroduplex formed between the sgRNA and the target strand.

sg RNA (gold); target DNA (green);