The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems provide bacterial and archaeal defense against mobile genetic elements.
• Well-studied types I, II, and III CRISPR-Cas systems use distinct proteins for crRNA maturation and nucleic acid interference.
• Different types employ specific nucleases like Cas6, Cas5d, RNase III, and Cas9 for RNA or DNA cleavage.

Purpose of the Study:

• To demonstrate a novel mechanism in CRISPR-Cas immunity.
• To characterize the dual-nuclease activity of type V-A Cpf1 from Francisella novicida.
• To elucidate Cpf1's role in both crRNA biogenesis and target DNA interference.

Main Methods:

• Investigated the enzymatic activities of Cpf1.
• Analyzed Cpf1's processing of precursor CRISPR RNAs (pre-crRNAs).
• Examined Cpf1's DNA cleavage mechanism, including target recognition and protospacer adjacent motif (PAM) interaction.

Main Results:

• Cpf1 acts as a dual-nuclease, essential for crRNA maturation and target DNA cleavage.
• Cpf1 cleaves pre-crRNAs upstream of hairpin structures, generating mature CRISPR RNAs (crRNAs).
• Cpf1 introduces double-stranded DNA breaks with a 5' overhang, guided by crRNA and recognizing a 5'-YTN-3' PAM.

Conclusions:

• Type V-A Cpf1 represents a novel class of enzymes with dual endoribonuclease and endonuclease activities.
• Cpf1-mediated immunity is the most minimalistic CRISPR-Cas system identified to date.
• This discovery expands the known diversity and mechanisms of CRISPR-Cas adaptive immunity.