A Complete Guide to CRISPR Cas9

Heralded as a pivotal moment in science, CRISPR is the game-changing human gene editing tool that has scientists the world over excited about  its potential to easily re-engineer biological systems and organisms. 

A genome is comprised of sequences of DNA that have specific roles , which are essential for biological functions. The process of genome editing involves changing the instructions of DNA sequences by cutting the sequence and tricking the cells into introducing a new message. Once a DNA sequence is cut, its natural repair systems will kick  in to fix the broken DNA strands. Effectively, scientists have the ability to edit any DNA template (mutagenesis) to alter how a gene functions.

Why not have a look at one of our other guides? Guide to temperature controlled logistics.

Currently, CRISPR is the most inexpensive and precise method of genetic manipulation on offer. 

Check out this video on the market behind gene editing, presented by Dr Ping Zhang of Oxford University.

CRISPR, which stands for “clustered regularly interspaced short palindromic repeats,” is regarded as one of the most important scientific innovations in recent years. In a nutshell, CRISPR  enables scientists to edit, modify and recombine any piece of genetic information in a way that is not only incredibly precise but comparatively inexpensive as well. Scientists claim that CRISPR makes gene editing a process that is no more difficult than editing a piece of code.

How does CRISPR work?

CRISPR is a naturally occurring system that comes from bacteria cells . It is essentially refers to DNA sequences that work to protect its host by identifying threats and attacking them, threats such as viruses.

When a host is threatened by a virus it has no innate way of protecting itself or fighting the virus. This is a process that has to be learned. CRISPR sequences work by storing strands of DNA from the threat, so it can recognize them later when the threat appears again.

At its core, CRISPR technology is comprised of two molecules that complete the process of introducing a mutation into a DNA sequence. These are Cas9 and RNA.

CRISPR predominantly refers to CRISPR-Cas9, a complex collection of enzymes that can find and edit DNA. CRISPR alongside the use of the Cas9 protein empowers scientists to edit a specific section of a gene sequence by deleting and replacing it with a new element.

Cas9 (CRISPR associated protein 9) is the enzyme that does the heavy lifting and introduces the mutation. It effectively acts like a pair of molecular scissors and it can cut a piece of the genome so that additional DNA can be added or removed.

RNA is the cellular mechanism used to guide Cas9. Synthetic RNA is tailored for Cas9 binding and coding, giving it the ability to identify the target genome that needs to be modified.

For a full list of CRISPR-Cas9 terms, check out this up to date glossary.

While CRISPR is predominantly used to defend the host from threats, scientists have learned that Cas9 can be instructed to carry out any number of tasks, whether that’s turning on a gene, shutting down a gene and more. The result is a tool that is excellent for editing DNA in a precise and inexpensive way.

SEE ALSO: Introducing The New Molecular Scissors - CRISPR & Cas9

Background

CRISPR certainly has a lot of hype surrounding it, but how did it all begin?

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Prior to gene editing, altering DNA was a much more labor-intensive process. A common method relied on radiation and chemicals to force mutations. However, this method was difficult to control, especially the location in which the mutation would appear.

While traditional methods of genome editing have yielded important discoveries in recombinant DNA, CRISPR gene editing stands out as the most efficient and cost-effect method. CRISPR-Cas9 and genome engineering research fields are two fields which merged in 2012 with the discovery that Cas9 can be reprogrammed by RNA and directed to cut any part of a DNA sequence. 

Key Developments

• In 2005, the Cas-9 protein was discovered by Alexander Bolton, of the French National Institute for Agricultural Research. 
• In 2012 Jennifer Doudna, a biochemist at UC Berkley discovered that Cas9 can be guided to a target.            
• In 2013, a report by Dr. Feng Zhang demonstrated that CRISPR-Cas9 can be used to correct genetic defects in plant cells .
• In 2015, CRISPR-Cas9 technology was heralded by Science Magazine as one of the most important technological advances in science in the last few years. 

A current high profile patent battle is ensuing between UC Berkeley‘s Jennifer Doudna (et al) and the Broad Institute of MIT’s Dr. Feng Zhang (et al), over who governs the IP concerning CRISPR. Both groups have made key discoveries and contributions to CRISPR, meaning that billions in funding and even a Noble Prize are up for grabs.

Uses for CRISPR

The excitement over CRISPR can be demonstrated by the sheer amount of attention it has gotten from leading companies and industries, including energy, manufacturing, agriculture, and pharma.

CRISPR has countless applications that that will greatly help with everyday life. For one thing, it has the potential to correct genetic defects, fight against disease, halt the spread of disease, cure genetic defects, grow climate-resilient crops, and help the development of pharmaceuticals and much more.

SEE ALSO: Cutting Edge CRISPR Cas9 Applications                

Cancer, in particular, is receiving a lot of attention in regards to CRISPR. Studies have attempted to alter damaged DNA from adults afflicted with cancer. It also has the potential to help combat medical conditions such as cholesterol, cystic fibrosis, cataracts and more.

Pharmaceuticals: Drug discovery and drug research

Human gene editing is a crucial aspect of the development of pharmaceuticals. By deliberately activating and deactivating certain genes, scientists can identify the proteins and genes that can either prevent or cause disease. These genes can then be the target of pharmaceuticals. It may even lead to personalized medicine.

Efforts are being led by CRISPR Therapeutics  working to develop transformative gene-based medicines for patients with serious diseases.

In addition, CRISPR has made it easier to mimic diseases, giving scientists more insight into the viability of a drug before it goes to clinical testing.  

Regulations and dangers

CRISPR is unique as it doesn’t use plant pathogens to edit DNA. Because of this, CRISPR won’t be regulated as a genetically modified organism. This development has cleared the way for the production of produce that can be made with CRISPR techniques, which may result in goods that have a longer shelf life or enhanced flavor.

Editing germline cells (reproductive cells) is currently illegal in the UK and many other countries. That said, editing somatic cells (non-reproductive cells) is a practice that is heavily encouraged and researched.

Challenges and risks

Although CRISPR-Cas9 has vast potential and the ability to provide solutions to some of our most pressing issues, it does have some challenges and risks that are inherent in its nature.

There are many challenges associated with CRISPR-Cas9. Some of them are; the ability to maintain end control on genetic manipulation, avoiding off-target effects, avoiding double-strand breaks, and ethics.

• Maintain end control of genetic manipulation:  Genetic mutations may be rejected as they are foreign proteins. Issues that may arise in human genetic manipulation are not fully understood. 

• Avoiding off-targets effects: One of the most prominent obstacles facing CRISPR is troubleshooting and the issues that may arise when the tool misses its target. Off-targets are hard to detect without whole genome sequencing and concerns have arisen over its use in a therapeutic setting. Fortunately, the market has opened the doors to new technologies that compliment CRISPR-Cas9 which help alleviate some of its shortcomings.

• Pathway recover post double-strand break: A double-strand break is one of the worst DNA lesions. Cells utilize two main pathways for self-repair, homologous recombination (HR) and no homologous end-joining (NHEJ). When a double strand break occurs, repairs may be error-prone, leading to further issues.

• Ethics: Additional areas of concern are more ethical in nature. Not only are there safety concerns when using CRISPR-Cas9 in humans, but there is the matter of undesired mutations which may cause havoc.

Additionally, the very notion that humans have the right to edit other biological issues is also an area of debate. For example, if CRISPR-Cas9 was used to fight malaria it may result in new and unknown diseases and viruses that are a danger to the public. Any long-lasting impact on biodiversity and ecosystems is also an area of concern that has to be considered.

SEE ALSO: Malaria: Scientists urged to not underestimate CRISPR’s dangers

The way forward

CRISPR and human gene editing is still in its infancy and faces many challenges. Researchers and scientists are currently competing to map out the treasure trove that is CRISPR, whether it pertains to searching for new bacteria to sequence;  there are a lot of variables which may affect its development.

That said, if it continues to receive even a fraction of the attention it is currently getting, we will see CRISPR as the solution to any problem that has a biological element. We may also see CRISPR-based produce and drugs hitting the shelves in the coming years.