Genome editing

Question 1

biotechnological use of genome editing techniques

Answer 1

CRISPR-Cas9​
TALENs​
ZFNs

Question 2

Zinc Finger Nucleases (ZFNs)​

Answer 2

FokI, a type IIS restriction enzyme has separate binding and cleavage activities ​

Nuclease is not sequence specific. Sequence specificity is engineered into binding domains ​

Each DNA binding domain recognises a 3 bp sequence – so in the example above, recognition of 18 bp (9+9) provides specificity. ​

ZFN (A) and(B) above may have different specificities.​

May be delivered on a plasmid or as RNA​

ZFNs are optimizable but it takes a lot of effort​

Question 3

TALENs​

Answer 3

Transcription activator-like (TAL) effectors (or TALEs) are made by Xanthomonas bacteria and are delivered by type III secretion system to the host cell​

TAL effectors provide specificity in DNA sequence recognition via variability in the Repeat Variable Diresidue (RVD) at positions 12 and 13 of a 33-34 amino acid sequence​

Can be used in a fusion protein with FokI nuclease – similar to ZFN​

Donor DNA (RNA) can be added for repair​

Question 4

Bacterial adaptive immunity uses Cas9​

Answer 4

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) used by bacteria and archaea​
CRISPR-Cas9 as a genome editing tool​
A guide RNA (gRNA) can be used to target any sequence for cleavage​
NHEJ means there is a possibility of INDELs (Insertion and deletion mutations)​
Efficiency of CRISPR-Cas9 >80%

Question 5

Precision engineering​

Answer 5

Cas9D10A is a mutant version of Cas9 which only retains DNA nickase activity​

​leaves only 1 strand and does not initiate NHEJ ​

Question 6

Inventive uses of CRISPR-dCas9​

Answer 6

Further mutation of Cas9 can also remove the nickase activity.​

This creates a protein which can be used to find any sequence on DNA​

Question 7

Biotechnological uses​

Answer 7

Stefan Jansson at Umeå University made the news by eating the first CRISPR meal​
“Tagliatelle with CRISPRy fried vegetables”.​
Uses in medicine and agriculture… what do you think?​

Question 8

CRISPR-Cas9 in more detail

Answer 8

CRISPR-Cas9 is a revolutionary genome editing technology that allows precise modification of DNA within living organisms. The system has its origins in the bacterial immune system and has been adapted for use in genetic engineering. Here’s a simplified explanation of how CRISPR-Cas9 works:

1. CRISPR Basics:
   CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats):
   Originally a bacterial defense mechanism against viruses.
   Bacteria store snippets of viral DNA (spacers) within their own DNA to remember past infections.
2. Adaptation:
   Spacer Sequences:
   If the bacterium survives a viral infection, it incorporates a small piece of the viral DNA (spacer) into its own CRISPR array.
   This acts as a genetic memory of the past infection.
3. CRISPR RNA Formation:
   Transcription:
   The CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA).
   Processing:
   The pre-crRNA is processed into smaller CRISPR RNA molecules, each carrying one spacer sequence.
4. Cas Proteins:
   Cas Proteins (CRISPR-associated):
   Enzymes responsible for recognizing and cutting DNA.
   Cas9 is the most commonly used enzyme in the CRISPR system.
5. Guide RNA (gRNA) Formation:
   Synthesis:
   A synthetic guide RNA (gRNA) is designed to be complementary to the target DNA sequence.
   It includes a sequence that matches the spacer from the CRISPR array.
6. Target Recognition:
   Binding:
   The gRNA guides the Cas9 protein to the target DNA sequence.
   Cas9 forms a complex with the gRNA.
7. DNA Cleavage:
   Recognition:
   The gRNA guides Cas9 to a specific location on the target DNA where it recognizes a specific sequence.
   Binding:
   Cas9 binds to the target DNA sequence.
   Cleavage:
   Cas9 acts as molecular scissors, creating a double-strand break in the DNA at the target location.
8. Repair Mechanism:
   Cellular Repair Systems:
   The cell’s natural repair mechanisms, such as Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR), are activated.
   NHEJ may introduce insertions or deletions (INDELs) during repair, leading to gene disruption.
   HDR can be harnessed for precise gene replacement or insertion if a donor DNA template is provided.
9. Applications:
   Genome Editing:
   CRISPR-Cas9 allows for precise modification of genes, enabling the addition, deletion, or replacement of specific DNA sequences.
   Functional Genomics:
   Used for studying gene function by disrupting or modifying specific genes.
   Therapeutic Potential:
   Holds promise for treating genetic disorders by correcting or modifying disease-causing genes.
10. Considerations:
    Off-Target Effects:
    One challenge is minimizing unintended modifications at off-target sites.
    Ethical Considerations:
    The power of CRISPR raises ethical questions, particularly regarding its use in humans and germline editing.
    CRISPR-Cas9 has transformed genetic research and holds enormous potential for medical applications, but ethical considerations and the need for precision remain critical aspects of its use. Researchers are continuously working to refine the technology for enhanced accuracy and safety.