4B PP - CRISPR-Cas9 in Bacteria

Bacteriophage

A bacteriophage is a virus that infects bacteria by inserting their genetic material (DNA or RNA) into the bacteria.

The bacteriophage uses the bacteria to replicate itself.

Bacteria have developed the CRISPR-Cas9 system to protect themselves against bacteriophages.

CRISPR-Cas9

genome.

Next time the virus invades, the bacteria transcribes the viral DNA and attaches it to Cas9 (an endonuclease).

The CRISPR array is a section of DNA with short, repeated sequences of nucleotide palindromes, that is:

 

Clustered Regularly Interspaced Short Palindromic Repeats 

The clustered repeats are interrupted by sections of viral DNA, called spacer DNA.

The CRISPR array is located downstream from the Cas9 gene

How does CRISPR-Cas9 work

The CRISPR-Cas9 system can be described in three steps:

 

1. Exposure

2. Expression

3. Extermination

CRISPR-Cas9 Exposure

1. The virus injects its DNA into the bacterium

2. Endonucleases come across the viral DNA and locate the Protospacer Adjacent Motif (PAM).

3. Endonucleases cut out a short section of viral DNA, known as the protospacer.
   
   Note: The PAM is not included in the final protospacer.

PAM

The PAM is a 2 - 6 nucleotide sequence that contains 2 consecutive guanine bases (NGG).

CRISPR-Cas9 Expression

1. The protospacer is inserted into the bacterium’s CRISPR array, becoming a spacer.

2. Upon re-infection by the same bacteriophage, the spacer is transcribed into an RNA molecule known as guide RNA (gRNA); a hairpin loop-like structure made up of:

3. gRNA binds to the Cas9 enzyme, creating a CRISPR-Cas9 complex.

Guide RNA (gRNA)

a hairpin loop-like structure made up of:

• A single RNA strand made from spacer and repeat RNA, called CRISPR RNA (crRNA).

• A single RNA strand made from another gene, called tracrRNA

CRISPR-Cas9 Extermination

1. CRISPR-Cas9 scans the cell for foreign viral DNA.

2. When CRISPR-Cas9 recognizes and binds to a PAM sequence, it separates the viral DNA double helix.

3. CRISPR-Cas9 checks if its gRNA is complementary to the viral DNA.

4. If the DNA is complementary to the gRNA, Cas9 cleaves the sugar-phosphate backbone, causing a double strand break (blunt ends).

5. CRISPR-Cas9 detaches from the DNA and continues searching the cell.
    
    
   
   

CRISPR-Cas9 Sum it Up

Exposure - Virus invades bacteria cell. Invading DNA from the virus is integrated into CRISPR array as a spacer.

Expression - production of crRNA and Cas protein. crRNA/Cas complex formed. The complex binds to invading DNA

Extermination - Degradation of viral genome

After CRISPR-Cas9

Once the viral DNA is cut, enzymes in the bacterium may naturally try to repair it.

However, these mechanisms are prone to errors, which can lead to mutations, such as nucleotide additions or deletions, rendering the viral DNA non-functional.

If a mutation does not occur, CRISPR-Cas9 will locate the gene again and repeat the process until the DNA repair mechanisms induce a mutation.