U3A1: 4B CRISPR-Cas9

Question 1

what is CRISPR

Answer 1

section of DNA with short, repeated and palindromic nucleotide sequences. it is found in bacteria for defence against viral attacks.

Question 2

what does CRISPR stand for

Answer 2

clustered regularly interspaced short palindromic repeats.

Question 3

explain the naturally-occuring process of CRISPR-Cas9

Answer 3

• bacterophage injects it’s DNA into bacterium.
• Cas1 and Cas2 endonucleases cut out short section of DNA known as a protospacer.
• protospacer introduced into bacterium’s CRISPR gene as a spacer.
• spacers transcribed into guide RNA (gRNA)
• gRNA binds to Cas9 to form CRISPR-Cas9 complex.
• CRISPR-Cas9 complex scans the cell for invading virus DNA complementary to gRNA.
• Cas9 cleaves sugar-phosphate backbone a few nucleotides down from PAM sequence.
• DNA will attempt to repair itself, however it is prone to errors and therefore mutations may deactivate it.

Question 4

explain the process of CRISPR-Cas9 for gene editing?

Answer 4

• synthetic gRNA is created complementary to the target DNA.
• Cas9 obtained with appropriate target PAM sequence.
• Cas9 and gRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex.
• gRNA-Cas9 mixture is injected into the specific cell
• Cas9 finds the target PAM sequence and checks that the gRNA aligns with the DNA.
• Cas9 cuts the selected DNA sequence using a blunt cut.
• the cell’s attempts to repair the break may silence the targeted gene.

Question 5

applications of CRISPR-Cas9

Answer 5

research:
- attaching fluorescent protein to Cas9 to locate specific genes
- disrupting gene expression to identify the function of specific genes.
dealing with disease:
- replacing deleterious alleles with healthy alleles
- adding genes that code for proteins which decrease susceptibility to infectious disease
- modify cancer-promoting genes to make them less influential
agriculture:
- pest and herbicide resistant genes
- altering genes to promote increased crop growth rates.

Question 6

ethical concerns of CRISPR-Cas9

Answer 6

• safety: potential for off-target cleavages and mosaics
• consent: embryos cannot give consent
• inequality: only the wealthy will be able to afford CRISPR technologies.
• discrimination: may threaten those judged by society as biologically inferior.