# Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum

## TL;DR

This paper introduces Fp2Cas9, a cold-adapted CRISPR enzyme that works efficiently at low temperatures, making genome editing possible in cold-water organisms.

## Contribution

The paper presents Fp2Cas9, a novel cold-adapted Cas9 nuclease with high activity at low temperatures and a refined PAM requirement.

## Key findings

- Fp2Cas9 cleaves DNA efficiently at 5°C with 75% activity, significantly higher than SpCas9 under the same conditions.
- The engineered sgRNA scaffold (sgRNA-V2) enables programmable DNA targeting for Fp2Cas9.
- In zebrafish, 2NLS-Fp2Cas9 achieved ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of embryos.

## Abstract

Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5′-SNAAAG-3′, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.