Note: Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

Note: Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

Doi: 10.1016/j.cell.2014.02.001

This paper presented the crystal structure of Streptococcus pyogenes Cas9.

Main domains:

• Target recognition

• Nucleases lobes (positively charge groove)

• HNH and RuvC nuclease domains

• Contain c-terminal which interact with PAM

CRISPR-Cas9 system has 3 types, namely I-III

• We used type II, cleaving DNA via the RNA-guided endonuclease Cas9

Cas9 nuclease composed of 2 domains;

• HNH -- cleave complement DNA strand

• RuvC -- cleave non-complement DNA strand

guide RNA (sgRNA) -- a synthetic fusion of crRNA (CRISPR RNAs) and trans-activating crRNA (tracrRNA) -- program to cleave any sequence preceding a 5’-NGG-3’ PAM sequence in mammalian cells.

Cas9: aa -- 1-1368 D10A/C80L/C574E/H840A (amino acid replacement to improve resolution) -- > in complex with 98 nt sgRNA, and a 23 nt target DNA

Replacement of amino acids does not affect the cleavage property as proved by cleavage property in HEK293FT.

Conformation between MolA and MolB are different but the DNA binding is similar. The crystal structure revealed the flexibility of the HNH domain of Cas9.

Cas9 consists of two lobes:

• A recognition (REC) lobe

• Long alpha helix (bridge helix)

• REC1

• REC2

• A nuclease (NUC) lobe

• RuvC

• HNH

• PAM-interacting (PI)

REC lobe

• Not share structural similarity with other known proteins

• Meaning -- Cas9-specific functional domain

• One of the least conserved regions across three Cas9 families within typeII CRISPR system (IIA, IIB, IIC)

PI (PAM interacting domain

• Adopt a novel protein fold

• Unique to the Cas9

RuvC domain

• Contain RNase H fold (non-sequence-specific endonuclease enzymes)

• Cleave the non-complementary strand of target DNA

HNH domain

• Has three catalytic residues (Asp839, His840,Asn863)

• Cleave the complementary strand of target DNA

• His840 is the critical residue to cleave complementary strand

sgRNA

• Tetraloop and stem loop 1 are critical for Cas9 function

BH domain (bridge helix domain)

• Tetraloop and stem loop 1 are critical for Cas9 function 

• Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78