• What is CRISPR-CasN?
• CRISPR-CasN enzymes
• Mechanisms of action
• Applications
• General procedure
• Delivery methods
• Why ribonucleoprotein?

CRISPR-Cas systems are classified into several types and classes based on their protein components and their mechanisms of action. There are six major types (Type I, II, III, IV, V, and VI) and multiple subtypes within each type, based on the presence of specific Cas proteins and their respective functions. Here's a brief overview:

• Class 1 CRISPR-Cas systems: These systems are characterized by multi-subunit effector complexes that consist of multiple Cas proteins. Class 1 CRISPR-Cas systems are further subdivided into three types:
  • Type I These systems have a large multi-subunit effector complex composed of multiple Cas proteins, including Cas3, Cas5, Cas6, and Cas7, among others. Type I systems are known for their RNA-guided DNA cleavage activity.
  • Type III These systems also have a multi-subunit effector complex composed of multiple Cas proteins, including Cas10, Cas7, and Cas5, among others. Type III systems are known for their RNA-guided RNA cleavage activity.
  • Type IV These systems are relatively rare and are characterized by a small effector complex composed of a single Cas protein. Type IV systems are known for their RNA-guided DNA cleavage activity.
• Class 2 CRISPR-Cas systems:

  These systems are characterized by a single-subunit effector protein that plays a key role in target recognition and cleavage. Class 2 CRISPR-Cas systems are further subdivided into three types:

  • Type II These systems are the most well-known and widely used for genome editing. They are characterized by the Cas9 nuclease, which requires both tracrRNA and crRNA for target DNA recognition and cleavage. The tracrRNA and crRNA can be artificially linked to use as a single guide RNA (sgRNA)

  • Type V These systems are characterized by the Cas12 nuclease, also known as Cpf1. Cas12 requires a single guide RNA (crRNA) for target DNA recognition and cleavage. Different from the blunt end of Cas9, Cas12a results in 5’ overhang sequences.

  • Type VI These systems are characterized by the Cas13 nuclease, which targets and cleaves RNA molecules, including RNA viruses. Cas13 requires a single guide RNA (sgRNA) for target RNA recognition and cleavage.

Classification and modular organization of CRISPR-Cas systems (Redrawn from Makarova et al., 2019. Nat Rev Microb)