Week 2 HW: DNA Read, Write and Edit

1. First, I created an account on Benchling. Then, I uploaded the Lambda sequence and introduced the restriction digestive enzymes. By combining them, I obtained the following result:

Lambda digestion Lambda digestion

2. Unfortunately, I did not have access to a laboratory equipped with all the necessary materials to perform the experiment.

3.

3.1 The human oxytocin (OXT) gene is located on chromosome 20 and encodes a precursor protein (prepropeptide) of 125–126 amino acids, which is subsequently processed into the active 9-amino acid hormone (nonapeptide: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly) and neurophysin I. The cDNA sequence for the human OXT precursor (NM_000915.4) consists of a 378-base pair open reading frame.

I used UniProt to identify the oxytocin sequence:

MAGPSLACCLLGLLALTSACYIQNCPLGGKRAAPDLDVRKCLPCGPGGKGRCFGPNICCAEELGCFVGTAEALRCQEENYLPSPCQSGQKACGSGGRCAVLGLCCSPDGCHADPACDAEATFSQR

3.2 Nucleotide Sequence

The nucleotide sequence of the human OXT gene coding region is as follows:

ATGGCTGGTCCTTCTCTGGCTTGCTGCCTGCTGGGCTTGCTGGCTCTGACTTCTGCCTGCTACATCCAGAATTGTCCGCTGGGCAAGCGGGCTGCTCCTGATCTGGATGTGCGGAAGTGTCTGCCTTGTGGTCCTGGGGGCAAGGGGCGGTGCTTTGGTCCTAATATCTGCTGTGCTGAAGAACTGGGCTGTGTTGGTACCGCTGAAGCTCTGCGCTGCCAAGAAGAAAATTATCTGCCTTCTCCCTGCCAGTCTGGCCAGAAGGCTTGTGGTTCTGGGGGTCGCTGTGCTGTTCTGGGCCTGTGCTGCTCTCCAGATGGTTGTCATGCTGATCCTGCTTGTGATGCTGAAGCTACCTTCTCTCAG

3.3 Codon Optimization for Oxytocin Expression

After determining the nucleotide sequence encoding oxytocin, codon optimization is necessary to ensure efficient expression in the chosen host organism. Although the genetic code is universal, different organisms prefer certain codons over others. This phenomenon is known as codon usage bias. If a gene contains codons that are rarely used in the host organism, translation efficiency may decrease, resulting in low protein yield or incomplete translation.

By optimizing codon usage, the DNA sequence is modified to incorporate codons that are more frequently used in the selected organism, without altering the amino acid sequence of oxytocin (CYIQNCPLG). This process improves translation efficiency, increases protein production, enhances mRNA stability, and reduces the risk of ribosome stalling.

4.1 I created an account.

4.2

Linear map Linear map

5.

5.1 (i) What DNA would I sequence and why?

I would sequence tumor DNA obtained from the patient as well as DNA from patient-derived organoids. The objective is to identify somatic mutations responsible for cancer development and progression.

Key cancer-associated genes that may be analyzed include TP53, KRAS, BRCA1, and EGFR.

Additionally, I would sequence germline DNA extracted from the patient’s blood in order to compare normal and tumor DNA. This comparison allows the distinction between inherited (germline) variants and tumor-specific (somatic) mutations.

The overall purpose is to identify actionable mutations and evaluate personalized therapeutic strategies using patient-derived organoids before administering treatment to the patient.

5.2 (i) What DNA would I want to synthesize and why?

I would synthesize a genetic construct encoding a tumor-specific CAR (Chimeric Antigen Receptor) or a CRISPR-based gene editing system that can be tested in patient-derived organoids.

Option 1: CAR construct (for personalized immunotherapy)

Target gene example: EGFR

Synthetic construct would include:

  • Promoter (e.g., CMV promoter)
  • scFv domain targeting tumor antigen
  • Transmembrane domain
  • Intracellular signaling domains (CD28/CD3ζ)
  • PolyA signal

Purpose:

  • Engineer immune cells or organoids to test personalized immunotherapy
  • Evaluate response outside the patient’s body
  • Optimize treatment before clinical use

Option 2: CRISPR therapeutic construct

Components to synthesize:

  • Cas9 coding sequence
  • Guide RNA targeting a mutated gene (e.g., TP53 mutation)

Target gene example: TP53

5.3 (i) What DNA would I want to edit and why?

In my project, I would edit tumor-derived stem cells or patient-derived organoids to:

  1. Correct oncogenic mutations
    • For example, restoring normal function of TP53.
  2. Knock out oncogenes
    • Such as mutated KRAS or EGFR.
  3. Introduce therapeutic modifications
    • Insert suicide genes
    • Enhance immune recognition
    • Increase sensitivity to chemotherapy

Goal:

  • Test gene correction strategies in organoids
  • Evaluate personalized therapies
  • Develop safer and more effective cancer treatments

5.3 (ii) What technology would I use?

I would use CRISPR-Cas9 genome editing technology.

CRISPR is:

  • Precise
  • Relatively simple to design
  • Efficient in human cells
  • Widely used in research and clinical trials

How does CRISPR-Cas9 edit DNA?

CRISPR-Cas9 works by creating a double-strand break (DSB) at a specific DNA sequence.

Essential steps:

  1. Guide RNA (gRNA) binds to the target DNA sequence.
  2. Cas9 enzyme creates a double-strand break.

The cell repairs the break via:

  • Non-homologous end joining (NHEJ) → gene knockout
  • Homology-directed repair (HDR) → precise gene correction using a repair template

Preparation and input

Design steps:

  • Identify target mutation
  • Design specific guide RNA
  • Design repair template (if correction is needed)

Input materials:

  • Cas9 protein or Cas9-expressing plasmid
  • Guide RNA
  • Repair template DNA (for HDR)
  • Patient-derived stem cells or organoids

Delivery methods:

  • Electroporation
  • Lipid nanoparticles
  • Viral vectors

Limitations

  1. Off-target effects
    • Cas9 may cut unintended DNA regions.
  2. Efficiency issues
    • HDR (precise correction) is less efficient than NHEJ.
  3. Mosaic editing
    • Not all cells are edited equally.
  4. Delivery challenges
    • Efficient and safe delivery into primary human cells can be difficult.

Conclusion

By using CRISPR-Cas9 to edit cancer-related genes in patient-derived organoids, we can:

  • Study mutation function
  • Test gene correction strategies
  • Develop personalized cancer therapies in a controlled ex vivo system