The applications of CRISPR gene editing technology are ever-expanding. A quick search of the latest publications mentioning CRISPR reveals uses in plant genome engineering, biosensing, genome screening, treating genetic diseases and diagnosing infections.

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

Since theinitial discoveryof what would become known as CRISPR,已经确定了6种主要类型和22个亚型CAS蛋白。

Perhaps the best known is Cas9 – the original gene editing enzyme whose transformative programmable capabilities were first described in 2012 byEmmanuelle Charpentier and Jennifer Doudna。Cas12 is a more recent addition to the CRISPR pantheon, gathering particular interest for its诊断的潜在用途。

But what are the major differences between Cas9 and Cas12, and how can you decide which is right for your research?

Introducing Cas9: the original CRISPR system

The Cas9 protein is found in several strains of bacteria including Streptococcus pyogenes，其中双RNA引导的DNA核酸内切酶最初进化为减少入侵的外源DNA。

In nature, 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 needed for the proper assembly and association with Cas9.

In their地标纸，Charpentier和Doudna的团队将这两个RNA结合在一起，将CAS9指导以在DNA中进行序列特异性切割。

他们还表明，可以对引导RNA的核苷酸序列进行编程，以靶向任何DNA序列进行裂解，形成了继续赢得他们的基因编辑系统的基础诺贝尔奖in 2020.

当它达到目标时，CAS9在DNA中进行了双链切割，从而导致能够插入，删除或修改附近DNA的特定区域。

Since Cas9 was discovered in 2012, many further developments have been made, including variants with even greater accuracy.例如，CAS9-HF1（或“ hifi”）was designed to reduce non-specific contacts and has a reported on-target cutting accuracy rateabove 99.9%。还进行了优化尺寸的优化CAS9的较小版本被发现金黄色葡萄球菌in 2015. More than 1 kb smaller than the original Cas9 fromStreptococcus pyogenes,this smaller Cas9 can be packaged into a single adeno-associated virus (AAV) vector and could represent a useful alternative in applications where size matters.

CAS9offers high fidelity genome editing that is particularly useful for research and commercial applications. The technology has already been put to work in labs around the world, generating nearly 20,000 scientific publications and contributing to the development of novel products ranging from genetically modified crops and livestock to food additives and beauty products.

Cas12: A new addition to the CRISPR family

然而，CAS9is not the only Cas in town: in 2015 Cas12 was在AcidaminococcusandLachnospiraceaefamilies of bacteria。This newer Cas has been much touted – but how does it differ from Cas9?

CAS12是单个RNA引导的核酸内切酶，这意味着它可以处理自己的导向RNA，因此仅需要CRRNA才能靶向，这使其比CAS9更小。为CAS9或CAS12设计靶向CRRNA的能力非常简单，并且是这些系统在基因组编辑中的简单性和广泛使用的关键。

CAS9has been widely reported to have a slightly higher cutting efficiency than Cas12, but both generally eclipse other genome editing systems like TALENs and zinc fingers when it comes to cutting efficiency. Cas9 leaves a blunt end cut, while Cas12 leaves a 5’ overhang, but this would not appear to have a meaningful impact on the types of edits that can be accomplished.

一个重要的区别是CAS9和CAS12识别不同的PAM sequences- 靶向DNA的短片段旁边是所需的切割部位 - 对每个系统的整体效用都有广泛的影响。

CAS9requires 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”.

然而，most mammalian genes are GC-rich, meaning it’s not difficult to find the requisite GG for targeting Cas9 to a specific location. By contrast, 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.

但是，研究人员发现了CAS12的独特属性，该特性将自己适用于基因组编辑之外的另一种应用：非特异性切割单链DNA。这种能力已变成powerful tool for DNA diagnosticsby detecting small amounts of DNA from sources such as viruses and cancer cells.

While Cas12 might hold more potential for certain applications like diagnostics, it’s at a much earlier stage of development than Cas9 and may lack some of the accuracy needed for precision genome editing.

作为原始和最佳特征的CRISPR效应核酸酶，CAS9具有更长的研究历史，出版记录和更大的投资。因此，CAS9可以“开箱即用”，并且更适合需要准确性和可靠性的商业和研究应用程序，尤其是在哺乳动物应用中。

Table 1:CAS9vs Cas12 – what's the difference?

	CAS9	Cas12 (also known as Cas12a or Cpf1)
Discovered	2012	2015
CAS家庭类型	II型	类型V。
Size	1,000–1,600氨基酸	1,100–1,300 amino acids
PAM sequence	G-rich	T-rich
切割类型	Blunt, 3 bp upstream of PAM	Staggered, 18–23 bp downstream of PAM
RNAs needed	crrna+ tracrRNA (or single-guide RNA)	crrna
Major application area	Mammalian gene editing	非哺乳动物应用和诊断
Number of publications	Approx. 20,000	Approx. 1,000

澄清CAS9和CAS12专利情况

As a patented technology, any commercial use of CRISPR – from internal R&D all the way through to market – requires a licence from the patent holder(s). And here’s where it gets a little confusing.

CRISPR不仅由于其科学潜力而迅速获得了突出性，而且在Charpentier，Vienna大学和加利福尼亚大学（称为CVC）和Broad Institute之间，媒体上的各种专利纠纷引起了很多媒体的关注。在麻省理工学院。这两个群体都拥有各种CRISPR技术的知识产权（IP）权利，必须获得许可才能在商业上使用。

最近的裁决in the US in February 2022 came down in favor of the Broad team having priority of invention for the specific use of single-guide CRISPR/Cas9 systems in eukaryotic cells. This sparked dramatic headlines and social media chatter that might lead some to incorrectly believe that CVC no longer has any patent rights over CRISPR/Cas9 technology.

实际上，这项最新裁决已有no impact on any of CVC's granted US foundational patents它涵盖了CRISPR/CAS9在所有环境中的组成和用途，包括真核细胞，在包括欧盟，中国，日本和其他地方在内的80多个国家中拥有的真核生细胞。

CVC retains the rights for over 40 US patents covering a range of compositions and methods for CRISPR/Cas9 gene editing, and holdsEuropean patents对于CRISPR/CAS9的DNA修饰方法，涵盖了包括人类在内的脊椎动物动物的细菌，植物，动物和细胞中的使用。

Most commercial research and applications of CRISPR/Cas9 are therefore likely to need tostart with a CVC licence，并且还可能需要与广泛的许可证，具体取决于地理区域和特定用例。

By contrast, despite the recent furore around Cas9 patent rights, the licensing and patent landscape for Cas12 is even more complex and opaque. As of 2020, Cas12 is claimed in899 patent families全球及其使用的许可状况仍然存在争议，完全不清楚。

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

CRISPR technology has revolutionized the life sciences, opening up new possibilities for novel technologies that will help to change the world. In turn, this has triggered an understandable rush of commercial interest from companies ranging from nimble biotech start-ups through to larger established organizations. Selecting the right system to use and securing the appropriate licensing is a vital first step in exploring this new world of opportunity.