Communications biologyJun 5, 2025

How CRISPR-Casλ2 cuts target DNA and processes its guide RNA

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 25, 2026

Abstract

The cryo-electron microscopy structures of Casλ2 revealed five distinct functional states.

Casλ2 demonstrated nuclease activity in both mammalian and plant cells, indicating its potential for genome editing.
The study identified a unique ruler mechanism by which Casλ2 processes precursor crRNA to mature crRNA.
Dynamic domain rearrangements during Casλ2 activation were observed, providing insights into its molecular mechanisms.
Structural comparisons with Casλ1 and CasΦ highlighted both diversity and conservation among phage-encoded type V CRISPR-Cas enzymes.
These findings enhance the understanding of CRISPR-Cas nucleases and may aid in the engineering of CRISPR-Casλ-based genome-editing tools.

Simplified

RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.

Key numbers

16–18 nt

Optimal Spacer Length for Cleavage

Casλ2 cleaves DNA optimally with guide RNAs of this length.

16–18 nt

Spacer Length of Mature crRNA

Casλ2 processes to mature crRNA of this length.

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Funding

Competing interests

Competing interests: G.M., A.J.G., A.O., L.E.A., G.R.H., T.D., W.X.Y., D.R.C., D.A.S., and Z.M. are current or former employees and shareholders of Arbor Biotechnologies. Arbor Biotechnologies has filed patents related to this work. The remaining authors declare no competing interests.

PubMed

How CRISPR-Casλ2 cuts target DNA and processes its guide RNA

Abstract

Key numbers

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Funding

Competing interests

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Abstract

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Related papers

You found one interesting study. We’ll send the next 7.

What Lands in Your Inbox Each Week:

Recent issues from the crispr gene editing brief