CORE CONCEPTS

Here are the essential biological concepts and principles that form the foundation of the CRISPR kit. Delve into them to understand the workings of the kit and its underlying processes, especially if these concepts are relatively new to you.

BIOLOGICAL CONCEPTS

DNA

DNA, Deoxyribonucleic Acid, is the blueprint of life, holding the genetic instructions that guide growth, development, function, and reproduction of living organisms. DNA’s structure, a double helix, looks like a twisted ladder where the rungs are composed of pairs of nucleotides – adenine (A) with thymine (T), and cytosine (C) with guanine (G).

RNA

RNA, Ribonucleic Acid, much like DNA, is a chain of nucleotides playing a critical role in translating the genetic information from DNA into proteins – life’s building blocks. RNA, though, is single-stranded, are composed of nucleotide uracil (U) instead of thymine (T) found in DNA. Messenger RNA (mRNA), Transfer RNA (tRNA), and Ribosomal RNA (rRNA) are the three primary types of RNA working in unison to create proteins.

Protein

Proteins, the fundamental building blocks of life, are large, complex molecules vital for the structure, function, and regulation of the body’s tissues and organs. Formed by linking amino acids in a sequence determined by DNA, proteins perform a myriad of functions from providing structural support to acting as enzymes, facilitating transport, enabling cell signaling and communication, and aiding in defense against foreign substances. In our kit, we use a type of chromoprotein, which produces a protein that contains a pigment. Different chromoproteins have been characterized from Nature with various colors.

The Central Dogma

The Central Dogma outlines the fundamental flow of genetic information within an organism,
comprising:

1. DNA Replication: Cells create an exact copy of their DNA prior to division.
2. Transcription: DNA’s genetic instructions are transcribed into messenger RNA (mRNA).
3. Translation: mRNA guides the assembly of proteins in the cytoplasm.

This sequential process, from DNA replication to protein synthesis, is essential to life, driving the functionality of an organism’s cells.

Gene

A gene is a segment of DNA, housing specific sequences of nucleotide pairs that dictate the formation of proteins. These proteins are cellular workhorses, tasked with diverse functions like structure provision, regulation of processes, and facilitation of chemical reactions.

Gene Expression

If we regard genes as a language of life, gene expression is how this language is spoken. A language that cannot be spoken is meaningless to life. Gene expression is the process through which the instructions within our DNA are used to create proteins. Gene expression is a tightly regulated process, which ensures the appropriate genes are active at the right time, in the right cells. A standard gene expression process involves two steps: transcription and translation.

Substrate

A substrate is the specific chemical reactant an enzyme binds to. It attaches to the enzyme’s active site and undergoes a reaction to form a product. The substrate’s shape and chemical properties must complement the active site for proper interaction.

Enzymes

Enzymes are biological catalysts, primarily proteins, that speed up reactions by lowering activation energy without being consumed. They enable essential processes like metabolism and DNA replication, that occur via specific substrates. Most enzyme names end in “-ase.”

Active Site

The active site is where the substrate binds and catalysis occurs. It has specific chemical properties that ensure precise interactions, enabling the transformation of the substrate into the product.

Co-factor/Coenzyme

These non-protein helper molecules assist enzymes. Co-factors are metal ions (e.g., copper, magnesium, zinc), while coenzymes are organic molecules, often derived from vitamins (e.g., Vitamin C). They stabilize interactions and enable electron transfer.

Activation Energy

The minimum energy needed to start a reaction. Enzymes lower this barrier, enabling biochemical reactions to proceed at sustainable rates for life.

Tyrosinase

A key enzyme in melanin production, catalyzing tyrosine oxidation in the biosynthesis pathway. Deficiencies in tyrosinase can cause conditions like albinism.

Melanin

A pigment responsible for skin color and UV protection, found in animals, plants, and microbes. It forms through enzymatic reactions involving tyrosinase.

TECHNOLOGICAL CONCEPTS

Gene Editing

Gene editing is a powerful technique that enables precise modifications to an organism’s DNA, much like using molecular scissors. If we regard DNA as the blueprint text of life, gene editing is the process to alter the text and its meaning. Gene editing has powerful applications in numerous fields, including disease treatment, agriculture, manufacture of valuable organisms and compounds, environment, climate, and ecology.

CRISPR-Cas9 gene editing

CRISPR, or “Clustered Regularly Interspaced Short Palindromic Repeats,” is a naturally occurring bacterial defense system against viruses, repurposed by scientists for gene editing. This system comprises two components: Cas9 protein and guide RNA (gRNA). The gRNA, akin to a mailman, precisely directs Cas9 to the specific DNA segment targeted for modification.

Inside a cell, Cas9 and gRNA collaborate to locate and cut the target DNA sequence, triggering the cell’s inherent repair mechanisms.

Two main repair pathways can be leveraged: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ can cause small sequence alterations, ideal for gene disruption or inactivation, while HDR, given a DNA template, facilitates precise DNA sequence alterations or insertions at the targeted site.

Thus, CRISPR gene editing represents a powerful tool for advancing biological understanding and potential health benefits.

CRISPR-dCas9 gene modulation

CRISPR-dCas9 uses a modified Cas9 protein, “dead” Cas9 (dCas9), to modulate gene expression without altering the DNA sequence. It acts as a switch button on genes, toggling genes on or off.

dCas9 can bind to specific DNA sequences guided by a complementary guide RNA (gRNA) but lacks the cutting ability of the original Cas9 protein. This binding prevents the normal genetic machinery from reading and activating the gene, effectively regulating its expression.

To modulate gene expression, dCas9 can be combined with additional components, such as activators or repressors. For example, we can attach proteins called activators to dCas9 to enhance gene expression, causing the gene to be turned on. Conversely, we can attach repressor proteins to dCas9 to suppress gene expression, turning the gene off.

In this kit, we utilize CRISPR-dCas9 gene modulation to ‘knockdown’ the expression of different chromoprotein genes.