Question

Question 1: What was determined to be the function of the CRISPR loci in microbes?

Question 2:   Explain the differences in the roles of the cas7 and cas9 gene.

Question 3: What genetic material does CRISPR target?

Question 4: Due to the ability of CRISPR to cleave DNA sequences at specific sites, it is considered a programmable version of what?

Question 5: Define and explain the significance of the PAM sequence.

Question 6: What is the role of tracrRNA in CRISPR function?

Question 7: What are the necessary components of the CRISPR-Cas9 interference system?

Question 8: What is the minimum number of nucleotides needed for crRNA to achieve effective cleavage?

Question 9:   Define and explain the process of the two genome editing strategies that were used before the emergence of CRISPR.

Question 10: What are potential ethical concerns with the use of CRISPR?

Question 11: What are possible future applications of CRISPR technology? (hint: solving environmental issues, medical issues, etc.)

Answer 1

Question 1: What was determined to be the function of the CRISPR loci in microbes?

Answer: CRISPR- Abrreviated as Cluster Regularly Interspaced Short Palindromic Repeats.

CRISPR loci in Microbes have several roles few mentioned below.

• They protect bacteria against phage infection
• Offer specific immunity like adoptive immunity im archea and bacteria providing resistance against invading genetic elements/foreign genetic elements
• CRISPR loci can be Used as template for strain genotyping to establish phylogenetic relationship and to identify historic path of a strain.
• Also can be used for epidmiological survey

Few research studies about CRISPR Loci

The CRISPR study in S.aureus helped in deiscovering new idea of preventing

Question 2:   Explain the differences in the roles of the cas7 and cas9 gene.

Cas7	Cas9
Cas7 class proteins belongs to type-III CRISPR System	It is Csn-1 a CRISPR associated protein
Cas7 is responsible for binding and processing of Cr-RNA(CRISPR RNA)	Naturally occurring genome editing system in bacteria
Possess a single RRM(RNA Recognition motif)	RNA programmable method to cleave DNA
Cas7 proteins form backbone of effector complexes protecting crRNA guide sequence	Allows permanent modification of genomic target sequence
These are active RNases required for Pre-crRNA cleavage	Can perform DNA repair
Cas7 along with Cas5 form clamps that secure the bound crRNA	Has a potential to be used for treatment of different disease like cancer, autoimmune disorders

Question 3: What genetic material does CRISPR target?

Answer: It targets DNA using short RNA

Question 4: Due to the ability of CRISPR to cleave DNA sequences at specific sites, it is considered a programmable version of what?

Answer: RNA programmable DNA endonuclease

Question 5: Define and explain the significance of the PAM sequence.

1. PAM- Protspacer Adjacent Motif
2. It is short DNA sequence made of 2-6 bp
3. essential for cleavage by Cas nuclease
4. found downstream of target DNA, targeted by guide RNA and cuts 3-4 bp by Cas nuclease upstream of it

Question 6: What is the role of tracrRNA in CRISPR function?

Answer:

1. tracrRNA- trans-Activating crRNA
2. maintains Cas9 in active form, allowing it to target DNA
3. Helps in binding of Cas9 to target DNA
4. helps in maturation of crRNA (CRISPR RNA)
5. tracrRNA complexed with crRNA called as gRNA very essential for DNA cleavage system.

Question 7: What are the necessary components of the CRISPR-Cas9 interference system?

Answer: CRISPR_Cas 9 requires three important components

1. Cas genes
2. leader sequence
3. repeat-spacer array

The immunity mediated by CRISPR-Locus involves three important process

1. Spacer aquisition
2. CRISPR RNA maturation
3. target Interference

Question 8: What is the minimum number of nucleotides needed for crRNA to achieve effective cleavage?

Answer; Minimum 17 nucleotides length is needed for crRNA fpr Cas9 to initiate cleavage system

Question 9:   Define and explain the process of the two genome editing strategies that were used before the emergence of CRISPR.

Answer: The two genome editing system used earlier were

1. Cre-loxP system:(CRE-Recombinas-loxP-locus of X-over P1)

It is specific recombination system used to target DNA sequence by external stimulus, this system used enzyme clled Cre recombinase for manipulating target DNA, it was used to carry out deletions, insertions, translocations and inversions of target DNA.

1. Flp-FRT system-Flp-Flippase-FRT-Flippase Recognition Target)

it is similar to Cre-loxP system, this system also uses site-directed recombination technology to manipulate DNA of a organisms by translocation and inversion, this system used flippase enzyme.

Question 10: What are potential ethical concerns with the use of CRISPR?

Answer;

1. Safety
2. Informed consent
3. Justice and equity
4. Genome-Editing Research involving embryos
5. Long term Side-effects
6. Regulations for consumers

Question 11: What are possible future applications of CRISPR technology? (hint: solving environmental issues, medical issues, etc.)

Answer: CRISPR can be used in future

1. for treating genetic disorders
2. For autoimuune disorders
3. to develop therapies that use gene editing to treat children's with sickle cell, hemophilia, cancer, blindness etc
4. To eliminate microbes that cause disease
5. To create healthier foods
6. To eradicate most dangerous pests
7. for Pet breeding
8. Can be used as DNA tape recorders
9. Decaf coffee bean
10. to discover greener fuels
11. used as a tool for de-extinction