CRISPR CAS 9 Flashcards
What is CRISPR Technology
A form of gene editing technology that targets a specific location on a gene and inserts, deletes, or replaces DNA at this location
What is CRISPR
A section of DNA containing short repetitions of nucleotides, involved in bacterial defence against viruses
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeat:
Clustered - positioned close together
Regularly - present at particular intervals
Interspaced - a space between two things
Short - measuring a small distance from end to end
Palindromic - a sequence that reads the same forwards as it does backwards
Repeat - present more than once
What does CRISPR do in Bacteria
CRISPR is an immune defence against genes that viruses inject into them to express as proteins and reproduce
So how does it function in bacteria?
: -> Upon infection by viruses, bacterial collect a chronological record of viral DNA which attacked them in CRISPR loci in the genome
- Transcription of CRISPR loci produces complementary mRNAs ( CRISPR RNA- CRNAs) that are complementary to viral DNA
- crRNAS form a complex with CRISPR associated with Cas 9 proteins which can cleave DNA
- crRNA guides the Cas9 to cut the viral DNA into fragments
- On infection by viruses, bacterial collect a chronological record of viral DNA which attacked them in CRISPR loci in the genome
- Cas 9 protein forms a complex with guide RNA in a cell
- This complex attaches to a matching genomic DNA sequence adjacent to a spacer
- Cas9-RNA complex cuts the double strands of DNA
What is a PAM region
- Protospacer adjacent motif (PAM) is found in viral genomes ( NOT bacterial genomes).
- It is a short DNA sequence ( 2-6 nucleotides ), usually denoted by a sequence ‘NGG’ ( N is any nucleotide).
- Is required for a CAS nuclease to cut and is generally found 3-4 nucleotides downstream from the cut
Cas Enzymes
- Restriction enzymes
- Can cut specifc regions of DNA found upstream of the PAM region
- Cas9 is a specific Cas endonuclease
Cas9
- Can cut at a specific region of the DNA, determined by a single guide RNA ( sgRNA)
- Has two active sites ( regions that can bind to DNA ) and is able to cut both strands of DNA at the sugar-phosphate backbone
What is a spacer sequence?
Spacer regions mean that next time the specific virus is encountered, the bacterium produces sgRNA, which directs Cas9 to cut the viral gene into pieces
What is Single Guide RNA
- Also known as sgRNA
- Is synthesised through transcription of the bacterials genome
- Is a combination of cRNA ( CRISPR RNA ) and tracrRNA (tracer RNA)
- sgRNA contains 20 nucleotides that are able to bind to a complementary sequence of a strand of DNA nucleotides after it unwinds the DNA
- sgRNA recognises the target DNA and directs the Cas9 enzyme there to cut
What is CRISPR RNA?
CRISPR RNA is transcribed from the spacer RNA (i.e the viral DNA that was inserted into the bacterial genome)
What is Tracer RNA?
Tracer RNA is also formed through transcription
Future of CRISPR-Cas9
The CRISPR-Cas9 system means that the sgRNA nucleotides can be built artificially
–> A scientist can design a sequence to match any specific sequence of nucleotides in DNA and control where in another organism’s DNA the Cas9 enzyme will cut.
–> Allows genetic engineers to remove a faulty allele and insert a normal functioning allele with extreme precision
–> Once target DNA has been cut by Cas 9, the cell’s natural DNA repair mechanisms act by doing both or either:
—> adding nucleotides that alter DNA sequence
—> Insert a short region of DNA with a specific nucleotide sequence
