Protein Modeling/CRISPR-Cas9

The CRISPR-Cas9 system and Anti-CRISPR proteins are the topic of Protein Modeling for the 2018-2019 season. The CRISPR complex known as Cascade can be found under the Protein Data Bank ID 4QYZ, and the Cas9 protein can be found under the ID 4OO8. The pre-build model is the anti-CRISPR protein AcrII4A, which can be found under the ID 5VW1.

CRISPR

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and it refers to a series of DNA sequences found in prokaryotes that help defend the organism from bacteriophages. In general terms, CRISPR stores Viral DNA separated by palindromic repeats, working as part of an active immune system in bacteria and archaea. CRISPR sequences are found in approximately 50% of bacteria and 90% of archaea that have been sequenced.

Cas Proteins

Proteins that merge with CRISPR to cut Viral DNA in bacteria. Several types and classes exist. See the Wikipedia Page on CRISPR for a detailed table of classes, types, and sub types of Cas.

Cas9

Large protein that cuts double-stranded DNA, works with CRISPR to form an active immune system in bacteria and archaea.

Cas1&2

This Cas protein takes viral DNA and adds it to CRISPR. See Mechanism for the overall process of the CRISPR-Cas9 system.

Important parts of CRISPR Cas9 System

PAM Sequence

Stands for protospacer adjacent motif. Short sequence downstream of target DNA. It is used to identify and locate the target DNA. Anti-CRISPR mimics this sequence to trick the CRISPR-Cas system.

crRNA&tracrRNA

1. crRNA(CRISPR RNA) is used to locate target DNA by binding to the PAM.

2. tracrRNA(trans-activating CRISPR RNA) - used to bind crRNA to Cas protein

CRISPR-Cas9 Mechanism

1. Spacer Acquisition - the Cas1 and Cas2 proteins will remove a 20-bp snippet of viral genetic material and add it to the bacteria's CRISPR array.

2. crRNA Biogenesis - CRISPR DNA is used to make crRNA, a "search tool" that finds the target Viral DNA

3. Target Interference - crRNA and tracrRNA form a complex with Cas9 and search through DNA until the crRNA "finds a match"(Detects a PAM sequence and the target DNA sequence). The Cas9 protein then makes a double stranded cut of the target DNA

The structure of the Cas9-dsDNA-sgRNA complex can be found in PDB file 5F9R.

Anti-CRISPR

Some phages use Anti-CRISPR(Found originally in Listeria Monocytogenes) to deactivate the CRISPR system. These proteins mimic the PAM sequence in DNA and fill the pocket in which the target DNA would fill in CRISPR-Cas9.

Inhibition of Cas9 by AcrIIA4(Anti-CRISPR)

Phages have also developed an evolutionary immune system to the CRISPR system. In a study published by Nature, Anti-CRISPR proteins were found to be highly acidic DNA mimics. (DOI: 10.1126/sciadv.1701620). In-depth studies by Yang & Patel and Dong, et. al provided insight into the structure and function of the AcrIIA4 protein. The main ways that AcrIIA4 inhibit the function of Cas 9 are:

• Blocking the CTD and Topo domains to prevent PAM recognition
• Blocking the RuvC domain to prevent cleavage of the non-complimentary strand

Cas9 must be in complex with an sgRNA in order for AcrIIA4 to bind to it. When AcrIIA4 binds to Cas9, the viral DNA cannot bind to the complex, which allows the virus to survive.

Structure

Cas9

The Cas9 protein has a bi-lobed structure, consisting of a REC (recognition) lobe and a NUC (nuclease) lobe. The lobes are further divided into domains. The REC lobe consists of 3 Helical domains and a Bridge Helix. The NUC lobe consists of a RuvC domain split into 3 parts, an HNH domain, a Topo domain, and a CTD domain. The RuvC domain includes an active site which cleaves the non-complimentary strand, and the HNH domain cleaves the complimentary strand. The Topo and CTD domains serve to identify the PAM sequence, as well as to bind to the non-complimentary strand. The Helical domains and Bridge Helix bind to the complimentary DNA strand.

AcrIIA4

The AcrIIA4 protein is a protein, with two chains. The B-chain consists of an alpha helix, a 3-stranded beta sheet, and 2 more alpha helices (N-C). The beta hairpins play a crucial role in the inhibition of Cas9, by occupying various active sites of the Cas9 protein.

Human Use

The crRNA and tracrRNA were found to be combinable in a single RNA strand called sgRNA(single guide RNA) by Jennifer Doudna. This single strand can be manipulated by scientists, giving scientists a quick and easy way to manipulate DNA(sgRNA can tell Cas where to bind and where to cut DNA). CRISPR-Cas9 rapid rise to popularity has been because of the relative ease and cost-effectiveness of using sgRNA and the system. Older technologies like ZFN and TALEN, especially ZFN, were time consuming to use.

Resources

• The official Center for BioMolecular Modeling page on the Protein Modeling event