What’s in a type? That which we call CRISPR, by any other name would…probably still edit genomes.
Understanding the different types of CRISPR systems can be as confusing as reading Shakespeare for the first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied interest in bacterial and archaeal immune systems. During this time, scientists discovered many related immune mechanisms that could target foreign DNA with unique properties. This led to a need to develop a classification system for the array of CRISPR technologies. Learning the general principles of this classification system will help you gain a deeper appreciation and understanding of CRISPR, and perhaps provide insights and ideas for your own research!
Classification Structure
Principles of CRISPR classification
While CRISPR systems now seem neatly organized into their respective categories, arriving at a consensus was not as straightforward as it seems. There is not a single gene that is shared across all CRISPR types; the fast evolution of these adaptive bacterial and archaeal immune systems has led to extreme sequence divergence that requires a multi-pronged analysis to sort effectively.
The current classification system relies on a combination of sequence similarity, phylogenetic analysis, neighborhood analysis of surrounding genes, and experimental data. In some cases, CRISPR systems that are in the same class and type don’t share sequence similarity but are grouped due to mechanisms of action and other analyses. This also means that between two types, the Cas effector may have the same name but be completely different! They are merely linked by a similar function.
After analysis, CRISPR classifications were placed into a fairly simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps to arrive at this hierarchy, the main aspects of each class and type are easy to understand. Classes are based on the main Cas effector; types are based on main function and composition; subtypes and variants are typically based on species of origin. There are currently two classes, six types, 33 subtypes, and 17 variants.
Class 1 versus class 2
There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use multi-Cas effectors, referred to as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out the function of the specific CRISPR type but often recruits the main effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2.
Class 1
Despite their incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas effector, Cascade. Developing tools using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar levels. It was only recently that researchers began building optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down into type I, type III, and type IV.
Type I
Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type I systems degrade large sections of DNA thanks to the dual action power of Cas3. Cas3 functions as both a helicase and a nuclease, unwinding and cutting up DNA after recruitment by Cascade. As you might imagine, this is handy for creating large genomic deletions. Type I systems are also employed as CRISPR transposases, by leaving out Cas3.
Type I is further subdivided into subtypes A–G. These subtypes have similar compositions and mechanisms, with the biggest differences being the components present in the Cascade complex.
Type III
Type III is considered the most complex of the CRISPR types and is thought to be the common ancestor of all other CRISPR systems. Type III systems are unique, as they can technically target both RNA and DNA, though it is largely believed that the cleavage of DNA is the primary function of this type of immunity. Within type III, there are six subtypes, A–F. All subtypes use Cas10 as the primary effector complex but are defined by the accessory Cas proteins in the Cascade complex.
Despite a tenuous grasp on how type III systems function, a III-E effector was developed, called Cas7-11. Despite being in Class 1, Cas7-11 is a single protein effector and is able to target and edit RNA in mammalian cells.
Type IV
Type IV is the middle child (like me!) of CRISPR types — weird and poorly understood. There are three subtypes, A–C, and they are grouped together because they contain a distinct Cas7-type gene. The exact mechanism of type IV systems is not known, but it’s suspected that they don’t carry out the same types of adaptive immune functions that other CRISPR types do, due to a lack of canonical CRISPR features (Pinilla-Redondo et al., 2019; Moya-Beltrán et al., 2021).
IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease effector. However, IV-C does in fact have a helicase domain due to a gene that resembles cas10. All type IV subtypes are missing adaptation genes, Cas1 and Cas2, which are critical for inserting short fragments of foreign DNA into the CRISPR array. Lastly, type IV systems are most often found on plasmids. The many unique features of IV-A and IV-B have led to the hypotheses that they are involved in plasmid competition or can hijack machinery from other CRISPR systems to carry out various functions.
Class 2
Class 2 CRISPR systems rely on a single protein Cas effector and are likely the class most people are familiar with. Class 2 CRISPR systems can be further broken down into type II, type V, and type VI, each relying on a unique Cas effector — Cas9, Cas12, or Cas13.
If you’d like to explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems called CasPEDIA, with information about enzyme activity, experimental conditions, and more.
Type II
Type II is the most popular and well-known type, as this is where Cas9 falls. The mechanism of type II is, unsurprisingly, well understood, and the first taught when introducing CRISPR as a technology. There are three subtypes, A–C, with the beloved SpCas9 (from Streptococcus pyogenes) belonging in II-A.
Cas9 was the first to be adapted for use in mammalian systems, with the landmark publication in 2013. Since that point, it has become the most engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, and more.
Type V
Type V uses Cas12 effectors and is one of the most popular alternatives to Cas9. Cas12 is unique because it can process multiple gRNAs under a single promoter, allowing for easy multiplexing. The double-stranded breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting edits using homology-directed repair. There are 10 subtypes of type V, named A–I and U.
V-A is the most popular Cas12 effector, but others have become more notable in recent years. A variant of V-U, paradoxically called V-K, is a CRISPR-associated transposase (CAST) that can insert large fragments of DNA. The V-F variants, which are among the smallest Cas enzymes (400 to 700 amino acids), use a Cas14 enzyme, and uniquely target single-stranded DNA non-specifically.
Type VI
Type VI is defined by the use of Cas13 effectors and are the only CRISPR type that exclusively targets RNA. Cas13 has become a natural successor to traditional RNA interference, as it can target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing. There are four subtypes, A–D, with VI-A, VI-B, and VI-D being the most common.
What’s your type?
It is highly unlikely that you will have to know and understand all the minute differences between the CRISPR types. However, knowing the basic diversity and the surface-level function of each type could lead you to new directions in your research. We all have our preferences. Your preferred type of CRISPR will likely depend on a variety of factors, including your goal, experience, hypothesis, and even the time and money available. The breadth and diversity of CRISPR systems will no doubt keep increasing, as efforts using large-scale clustering and discovery algorithms find new candidates for CRISPR proteins. A type VII is already on the horizon (Altae-Tran et al., 2023)!
References and Resources
References
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Additional resources on the blog
- CRISPR 101: Cas9 vs. The Other Cas(s)
- CRISPR 101: RNA Editing with Cas13
- Build your CRISPR vocabulary
Additional resources on Addgene.org
Topics: CRISPR, CRISPR 101
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