Topic 14: Crisper

Question 1

Crisper (2)

What it is+ derived

Answer 1

• technology that can be used to edit genes in plant and animal genomes
• derived from a bacterial immune system (bacterial defensive system)

Question 2

Steps for crisper (4)

Answer 2

1. acquire foreign DNA sequences (spacers) from viral infection (Cas1 and Cas 2 complex cleaves DNA from viral infection)
2. Add spacer to genomic CRISPER region “memory of what it has been affected with”
3. Transcribe the genomic entire CRISPER region of dna into guide RNA.
4. gRNA assembles with Cas 9 protein to guide cleavage of invading DNA

Question 3

Tell me nore about the Crisper Cas 9 system and how it works (4)

PAM x2

Answer 3

• we need both Cas9 enzyme and gRNA to be expressed in cell of interest
• gRNA will direct Cas9 to introduce a double stranded break at a percise location
• The target sequence must be 3 nucleotide upstream of the PAM (5’ NGG)
• Landing at PAM, Case 9 helps unwind sequence so gRNA can come in and Cas9 then cuts

Question 4

Double stranded break can be repaired by (2)

Answer 4

• homologous recombination
• Nonhomologous end joining

Question 5

Homologous recombination

Answer 5

Introduces donor DNA which is used as a template to repair the double stranded break. Complementary at ends to pair with cut ends.
uses an undamaged DNA template on the sister chromatid or homologous chromosome to repair the break, leading to the reconstitution of the original sequence

Question 6

Non homologous end joining

Answer 6

• A messy repair system
• introduced deletetions/mutations that can inactivate the gene
• NHEJ modifies the broken DNA ends, and ligates them together with little or no homology, generating deletions or insertions

Question 7

Non homologous repair vs homologous repair

Answer 7

NHDEJ which identifies any two broken ends of DNA and “sticks” them back together, the HDR pathway proteins recognize homologous sequences of DNA (from a sister chromatid, a donor homology plasmid, single stranded ODN, etc.) near the region of the DSB and uses those homologous regions as a template for the precise “correction” of the damage. Researchers can take advantage of this mechanism by flanking any DNA sequence of interest with “homology arms”, or regions of flanking homology to the DSB site. This allows geneticists to effectively insert any sequence of DNA (limitations) into the exact genomic loci as determined by your CRISPR cut sites and flanking homology arms.