Synergistic engineering of Casδ nuclease for robust genome editing - PubMed
4 hours ago
- #Genome Editing
- #CRISPR
- #Casδ
- Casδ is a compact CRISPR system (~900 amino acids) with broad temperature tolerance and uses short crRNA without tracrRNA.
- The low editing efficiency of Casδ in eukaryotic cells limits its application.
- A hierarchical engineering strategy was developed to improve Casδ-1's genome editing activity, focusing on interactions with crRNA, PAM duplex, single-stranded DNA, and RNA-DNA heteroduplex.
- An enhanced variant, enCasδ, was created with 9 amino acid substitutions, increasing editing efficiency.
- enCasδ showed 1.3- to 29.3-fold higher editing activity than wild-type Casδ-1 in human cell lines, with an average efficiency of 54.6%.
- In maize, enCasδ's editing efficiency increased by an average of 5.3-fold, reaching up to 80% at specific loci.
- enCasδ's performance is comparable to SpCas9 and other Cas12 nucleases.
- enCasδ broadens the applicability of the Casδ CRISPR system for robust genome editing in both animal cells and plants.