Can you choose Cas9 or Cas12 by choosing the right CRISPR system for your research?

Can you choose Cas9 or Cas12 by choosing the right CRISPR system for your research? ...

The use of CRISPR''s gene editing technology is becoming increasingly popular. A quick search of the most recent reports revealing CRISPR''s capabilities in plant genome engineering, biosensing, genome screening, treating genetic diseases, and diagnosing infections is underway.

A large part of the versatility of the CRISPR system comes from the CRISPR-associated protein or Cas protein, the molecular scissors that recognize and cut specific pieces of DNA.

Six major types and more than 22 subtypes of Cas proteins have been identified since theinitial discovery of what would become known as CRISPR.

Cas9 is the first gene editing enzyme to be able to transformative programmable abilities described in 2012 byEmmanuelle Charpentier and Jennifer Doudna. Cas12 is a more recent addition to the CRISPR pantheon, attracting particular interest for its potential use in diagnostics.

What are the major differences between Cas9 and Cas12, and how can you choose which one works best for your research?

Introducing Cas9: the original CRISPR system

The Cas9 protein has been found in several strains of bacteria, including Streptococcus pyogenes, where the two RNA-guided DNA endonuclease originally developed to reduce invading foreign DNA.

Cas9 requires two RNAs to recognize and cut its target a targeting RNA called CRISPR RNA (crRNA), which is a copy of the target DNA, and a structural component called trans-activating CRISPR RNA (tracrRNA), which forms a complex with crRNA that is required for proper assembly and association with Cas9.

Charpentier and Doudnas'' team grouped these two RNAs into a single guide RNA, which might direct Cas9 to make sequence-specific cuts in DNA in theirlandmark paper.

They also demonstrated that the guide RNA nucleotide sequence may be programmed to target any DNA sequence for cleavage, forming the basis of the gene editing system that continued to win the Nobel Prize in 2020.

Cas9 cuts a double-stranded segment in the DNA when it attains its target, making it a better choice, allowing for insertion, delete, or modify specific areas of the DNA nearby.

Many further changes have been made since Cas9 was discovered in 2012, including variants with even greater accuracy. For example, the asmaller version of Cas9 was designed to reduce non-specific contacts and has a reported on-target accuracy rate of 99.9%. Size optimizations have also been made, with this smaller Cas9 being discovered in Staphylococcus aureus in 2015. It can be transformed into a single adeno-associated virus (AAV) vector and might serve as a useful

Cas9 offers high-fidelity genome editing that is particularly useful for research and commercial applications. It has already been put to work in laboratories around the world, generating over 20,000 scientific publications and contributing to the development of novel products, from genetically modified crops and livestock to food additives and beauty products.

Cas12: A new addition to the CRISPR family

Cas9 is not the only Cas in town, but in 2015 Cas12 was discovered in the acidaminococcus and Lachnospiraceae families of bacteria. This newer Cas has been extensively discussed, but how does it differ from Cas9?

Cas12 is a single RNA-guided endonuclease, which means it process its own guide RNAs and thus only requires crRNA for targeting, making it a smaller overall package than Cas9. It is very simple and straightforward to use these systems in genome editing.

When it comes to cutting efficiency, Cas9 is widely reported to have a slightly higher cutting capacity than Cas12, but both may be superior to other genome editing methods, such as TALENs and zinc fingers. Cas9 leaves a blunt end cut, while Cas12 leaves a 5 overhang, but this would not imply a significant impact on the kinds of edits that may be made.

Cas9 and Cas12 recognize differentPAM sequences the short piece of targeting DNA next to the desired cleavage site, which has big implications for each system''s overall performance.

Cas9 requires only a GG sequence adjacent to its target, whereas the original Cas12 requires the sequence TTTV (where V is A, C, or G), with more recent derivatives of Cas12 requiring only TT.

The canonical Cas12 TT-rich PAM is more abundant in bacterial genomes and therefore has a more limited targeting capability in mammalian genomes. Based on this alone, many researchers find Cas9 much more flexible for use in mammalian systems.

Cas12''s unique feature has been discovered by researchers, proving itself to other applications outside of genome editing: non-specific cutting of single-stranded DNA. This capability has been made a powerful tool for DNA diagnostics by detecting small amounts of DNA from sources such as viruses and cancer cells.

Cas12 may offer enhanced capabilities for certain applications, such as diagnostics, but it is at an early stage of development than Cas9, and might not provide the exact exactness required for genome editing.

Cas9 is well-known for its research history, publication record, and higher investment. For this reason, Cas9 is a great way to go right out of the box, especially for commercial and research applications where accuracy and reliability are required, especially in mammalian applications.

What''s the difference between Cas9 and Cas12 in Table 1?

Cas9

Cas12 (also known as Cas12a or Cpf1)

Discovered

2012

2015

Cas family type

Type II

Type V

Size

1,0001,600 amino acids

1,1001,300 amino acids

PAM sequence

G-rich

T-rich

Cut type

Blunt, 3 bp upstream of PAM

Staggered, 1823 bp downstream of PAM

RNAs needed

crRNA + tracrRNA (or single-guide RNA)

crRNA

Major application area

Mammalian gene editing

Non-mammalian applications and diagnostics

Number of publications

Approx. 20,000

Approx. 1,000

Cas9

Cas12 (also known as Cas12a or Cpf1)

Discovered

2012

2015

Cas family type

Type II

Type V

Size of the screen

10001,600 amino acids

1,1001,300 amino acids

PAM sequence

G-rich

T-rich

Type of cutlery

Upstream of PAM, Blunt, 3 billion people

PAM was saggered in 1823, with a slew of tens of thousands of pounds downstream.

RNAs are required.

crRNA + tracrRNA (or single-guide RNA)

crRNA

A major application area

Genetic manipulation in Mammalians

Non-mammalian applications and diagnostics

The number of publications that are published

As many as 20,000 people have gathered in the country.

Over 1.000 people have been born in the United Kingdom.

Cas9

Cas12 (also known as Cas12a or Cpf1)

Discovered

2012

2015

Cas family type

Type II

Type V

Size

1,0001,600 amino acids

1,1001,300 amino acids

The PAM sequence is now available.

G-rich

T-rich

Cut type

Upstream of PAM, Blunt, 3 billion people.

PAM was damaged in 1823, and the industry has recovered.

RNAs are required.

crRNA + tracrRNA (or single-guide RNA)

crRNA

The major application area

Mammalian gene editing

Non-mammalian applications and diagnostics

The number of publications that have been published has increased.

20,000 people are expected to be aboard the XXX million dollar program.

Approx. 1,000 people have come to the utmost.

Clarifying the patent situation for Cas9 and Cas12

Any commercial use of CRISPR from internal research and development until to market requires a patent holder''s license. It''s where it''s a little confusing.

CRISPR has gained an equities in the field of technology, but there has been a lot of media attention for the various patent disputes over the technology between Charpentier, the University of Vienna, and the University of California (together known as CVC) and the Broad Institute at MIT. Both groups have intellectual property (IP) rights to several items of CRISPR technology that must be licensed to be used commercially.

The most recent judgment in the United States in February 2022 came down in favor of the Broad team giving the priority of invention for the specific use of single-guide CRISPR/Cas9 systems in eukaryotic cells. This sparked massive headlines and social media chatter that might result in some incorrectly believing that CVC no longer has any patent rights over CRISPR/Cas9 technology.

This latest measure has no impact on any of CVC''s foundational patents that cover the compositions and uses of CRISPR/Cas9 in all settings, including eukaryotic cells, which it has in over 80 countries, including the EU, China, Japan, and elsewhere.

CVC maintains the rights for over 40 US patents covering a wide spectrum of compositions and techniques for CRISPR/Cas9 gene editing, and holds European patents for the CRISPR/Cas9 method of DNA modification, aimed at bacteria, plants, animals, and cell from vertebrate animals.

Most commercial projects and applications of CRISPR/Cas9 are expected to begin with a CVC licence, and may also require a separate licence from the Broad, depending on the geographic area and the chosen use case.

Despite the recent outcry about Cas9 patent rights, the licensing and patent landscape for Cas12 is much more complex and opaque. As of 2020, Cas12 is claimed in 899 patent families worldwide, and the licensing situation for its use remains in jeopardy.

Several organizations have made public claims to have created Cas12 derivatives that are free and clear of restrictions, but these claims appear to be dubious at best given the structural similarities to Cas12 and the ever-increasing number of patents being filed in this space in hopes of getting a piece of the IP pie.

CRISPR technology has triggered an enticing rush of business interest from businesses ranging from small biotech startups to larger established organizations. Choosing the right system to use and obtaining appropriate licensing is a crucial first step in exploring this new world of possibilities.

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