CRISPR-Cas9 Gene Editing Is On The Cusp Of Something Big

 

Natali_Mis

Gene editing, also known as genome editing, is a method where the DNA of an organism is modified using biotechnological techniques. It allows scientists to add, remove, or alter genetic material at particular locations in the genome.

This 2-part series will cover the basics of CRISPR-Cas9 (see below) in part 1 and the major players involved and how to differentiate the companies competing in this space in part 2. I think it is an important time to learn about this technology now that we are on the cusp of the first approval in the space and are potentially coming out of what was a terrible 3 year bear market for biotechs.

CRISPR-Cas9: The most widely used and recognized gene editing technology is CRISPR-Cas9. It was developed into a genome editing tool by Emmanuelle Charpentier and Jennifer Doudna, who were awarded the 2020 Nobel Prize in Chemistry for their work. Feng Zhang and George Church also made significant contributions to its development for use in eukaryotic cells (like human cells).

CRISPR-Cas9 is a revolutionary gene-editing tool that many regard as transformational because of its simplicity and versatility. Many experts think it has the potential to completely revolutionize human health.

Here's a quick overview of how it works:

CRISPR-Cas9 Basic Parts

1. CRISPR Sequence: Originally part of the bacterial immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences are segments of DNA containing short repetitions of base sequences.
2. Cas9: An enzyme that acts like a pair of molecular scissors. It can cut DNA at a specific location.
3. Guide RNA (gRNA): A piece of RNA that is designed to be complementary to the target DNA sequence. It “guides” the Cas9 to the right part of the genome.

CRISPR-Cas9 Process

Designing the Guide RNA: Researchers design a gRNA that matches the sequence of the DNA they want to edit.

Designing a gRNA for CRISPR-Cas9 gene editing involves a precise process to ensure specificity and efficiency in targeting the desired DNA sequence. Here's an overview of how researchers design a gRNA:

Identifying the Target Sequence

Target Selection: Researchers first identify the specific sequence in the DNA that they want to edit.

Identifying the specific DNA sequence to edit using CRISPR-Cas9 or any other gene editing tool involves a combination of scientific knowledge, research, and bioinformatics tools.

Often, the desire to edit a specific DNA sequence stems from prior research. For instance, scientists might target a gene known to cause a disease or a genetic pathway influencing a particular trait.

Researchers review scientific literature to understand the function of genes and their associated sequences, relying heavily on databases like GenBank, the UCSC Genome Browser, and the ENSEMBL database which provide comprehensive genomic information for various organisms.

Advancements in computing power with tools like machine learning and artificial intelligence is helping speed up the research process. Software tools can analyze genetic sequences to identify genes and analyze prior clinical trials to determine target areas and chances of success faster and more accurately than ever before.

The target sequence should be unique to the region of interest to minimize off-target effects, where the CRISPR system edits unintended parts of the genome.

A burgeoning field of analytics has sprouted up to assist in determining off target edits. The nemesis of CRISPR-Cas9 and the entire gene editing field is off target edits. Regulators like the FDA in the USA are keenly focused on off target edits when evaluating the risk-reward of gene editing trials underway.

Documents published Friday revealed the agency plans to focus a meeting of expert advisers next week on this risk, known as “off-target” edits, and on whether there's enough evidence to show it's not a safety concern.

– Biopharma Dive, October 27, 2023

BioPharma Dive

PAM Sequence Requirement: They must also consider the presence of a PAM (Protospacer Adjacent Motif) sequence near the target site. Cas9 requires a short, specific DNA sequence right next to the target site for binding and cutting. The PAM sequence is essential for Cas9 binding and cleavage.

Designing the gRNA Sequence

• Complementary Design: The gRNA is designed to be complementary to the target DNA sequence, allowing it to bind specifically to that sequence. Typically, about 20 nucleotides of the gRNA are designed to be complementary to the target site. A nucleotide is the basic structural unit and building block of DNA and RNA – they consist of 3 components (nitrogenous base, sugar molecule, and phosphate group) and their role is to store the genetic information of an organism.
• Avoiding Off-Target Effects: As I mentioned before, it's crucial to ensure that the gRNA is specific to the target sequence and does not bind to other similar sequences in the genome, which can lead to off-target edits. Analytical tools are used to analyze the potential off-target sites.

Using Bioinformatics Tools

• Software Assistance: Researchers use various software and online tools to design gRNA sequences. These tools help in identifying the most efficient and specific gRNA sequences, taking into account factors like the efficiency of targeting, potential off-target sites, and the presence of the PAM sequence.
• Optimization: The tools also assist in optimizing the gRNA for various factors like GC content, which can affect the stability and effectiveness of the gRNA.

Synthesizing the gRNA

• Once the sequence is designed, the gRNA is synthesized. This can be done chemically, or the gRNA can be transcribed in vitro or in cells from a DNA template.

Testing and Validation

• After synthesis, the gRNA is tested in cells or in vitro to ensure it effectively targets the desired DNA sequence. This involves checking for the intended edits and assessing any potential off-target effects.

1. Formation of the CRISPR-Cas9 Complex: The gRNA binds to the Cas9 enzyme, forming a complex.
2. Targeting the DNA: The gRNA within this complex guides Cas9 to the target DNA sequence in the genome by base pairing with the complementary DNA sequence.
3. Cutting the DNA: Once the CRISPR-Cas9 complex locates and binds to the target DNA sequence, Cas9 cuts the DNA at this precise location. This cut typically happens in both strands of the DNA helix, creating a double-strand break (DSB).
4. DNA Repair and Editing: After the DNA is cut, the cell's natural repair mechanisms kick in. There are two main pathways for repair:
   • Non-Homologous End Joining (NHEJ): This pathway often introduces insertions or deletions (indels) at the cut site, which can disrupt the gene, effectively “knocking out” its function.
   • Homology-Directed Repair (HDR): If a DNA template with a desired sequence is provided, the cell can use this template to repair the cut, incorporating the new sequence into the genome. This allows for precise gene editing, such as correcting a mutation.

5. Experimental Validation

• In Silico Analysis: Before actual experiments, in silico (computer-based) analysis predicts the effectiveness and specificity of the target site.
• Laboratory Testing: Initial experiments in cell cultures or model organisms help validate the target site's effectiveness and safety.

CRISPR Applications in Human Health

The application of CRISPR in human health is creating treatments for genetic disorders by correcting genetic defects. Just like a computer program with faulty coding, a human with a faulty genetic code can now be edited. the advantage is CRISPR has high precision, is relatively easy to design and implement, and is adaptable to many different organisms.

The challenge for CRISPR is there are genuine ethical concerns, particularly regarding human germline editing. This is editing done to a human that can be passed on to subsequent generations. Any genetic changes made in these germline cells will be passed on to all cells of the resulting organism, including its own germline cells. This means the changes can be inherited by the organism's offspring.

There are also potential off-target effects (unintended edits in the genome); and the efficiency and precision of the editing process is still be sussed out.

Who are the Major CRISPR Companies that are Publicly Traded?

In part 2, we will discuss the major players in CRISPR that you can invest in right now and differentiate amongst them. But for now, these are the major CRISPR companies that are publicly traded:

1. CRISPR Therapeutics (CRSP): co-founded by one of the pioneers of CRISPR-Cas9, Emmanuelle Charpentier. It focuses on developing gene-based medicines for serious diseases. CRISPR is on the cusp of potentially having the first FDA approved therapy using gene editing, CASGEVY (exa-cel) – the FDA rules on this on December 8, 2023. CRSP has roughly $1.4B in net cash vs annual cash burn of $350 Million so it sits in a good position as it waits for its first drug to be approved. CRSP also will get $200M from its partner Vertex Pharmaceuticals (VRTX) if/when exa-cel is approved. CRSP stands to receive 40% of revenues from VRTX if their drug is approved.

2. Editas Medicine (EDIT): This company is engaged in discovering and developing genome editing therapies. It works on treating patients with genetically defined diseases by correcting their disease-causing genes.

3. Intellia Therapeutics (NTLA): Intellia is known for its work in developing in vivo (inside the body) gene editing treatments. It aims to use CRISPR-Cas9 technology to treat various diseases, including rare genetic conditions.

4. Beam Therapeutics (BEAM): Beam Therapeutics stands out for its focus on base editing, a more precise form of CRISPR editing that allows for single base pair changes without creating double-strand breaks in DNA.

5. Caribou Biosciences (CRBU): Co-founded by Jennifer Doudna, a CRISPR-Cas9 co-inventor, Caribou Biosciences focuses on CRISPR genome editing technology to develop cellular immunotherapies for various diseases, including cancer.

6. Prime Medicine (PRME): Prime Medicine's approach represents an advancement in the field of gene editing, particularly in providing solutions for a wide spectrum of diseases through precise genetic modifications. Their work in Prime Editing holds promise for addressing genetic conditions that were previously challenging to treat.

I hope this covers the basics of CRISPR-Cas9 and gene editing. This revolutionary tool has the potential to disrupt health care and make a lot of wealth for investors that choose the correct companies to invest in.