4B CRISPR-Cas 9

• CRISPR is a naturally occurring sequence of DNA found in bacteria that plays an important role in their defence against viral attacks.

  It is a section of DNA with short, repeated sequences of nucleotides that have the same forward and reverse read.
• CRISPR-Cas9 is a complex which can cut a target sequence of DNA (creates blunt ends at a site specified by gRNA)
• Bacteriophage is a virus that infects prokaryotic organisms, such as, bacteria.
• CRISPR stands for Clustered Regulary Interspaced Short Palindromic Repeats
• Spacer is the viral 'mugshot' of DNA cut from invading bacteriophages, it allows recognition during future following/subsequent invasions.

  The clustered repeats get interrupted by spacer DNA.
• Steps in CRISPR-Cas9 defence system:

  1. Exposure
  2. Expression
  3. Extermination
• Exposure: Bacteriophage injects its DNA into a bacterium, the bacterium identifies the viral DNA as foreign. Cas1 and Cas2 enzymes cut out a short section of viral DNA (around 30 nucleotides), called a protospacer.

  The protospacer is introduced into the bacterium's CRISPR gene, becoming a spacer.
• Expression: CRISPR spacers are transcribed along with half a palindrome/symmetrical from the repeat on each side. This transcription creates a gRNA which is an RNA molecule. gRNA binds to Cas9 to form a CRISPR-Cas9 complex. The complex targets viral DNA within the cell that matches the gRNA. gRNA forms a hairpin loop structure from the transcribed palindromic repeats adjacent to the space.
• Extermination: CRISPR-Cas9 complex scans cell for matching viral DNA. Cas9 cleaves DNA backbone to deactivate virus. Cas9 has two active sites for cutting both DNA strands creating blunt ends.
• gRNA (guide RNA): RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific site.