Prime editing enables the precise modification of genomes, thereby holding great potential for the treatment of genetic diseases. Despite substantial advancements in prime editing technology and the initiation of the first clinical trial for treating chronic granulomatous disease, further enhancement of editing efficiency across edit types is still urgently needed. Here, we developed a compact prime editor, PE2ΔR, by deleting the RNase H domain of the MMLV reverse transcriptase (MMLV-RT). We then conducted a saturated mutagenesis screen targeting two DNA interacting regions within the PE2ΔR-RT Fingers domain. By integrating three highly effective mutations (I61R, V101R, S67W) into PEmax lacking RNase H domain (termed PEmaxΔRM3), we achieved up to a 90% increase in editing efficiency across editing types compared to PEmax. Structural modelling using AlphaFold 3 suggests that these mutations enhance primer–template stabilization and guide the RNA/DNA hybrid into a catalytically favourable trajectory, providing a mechanistic explanation for the enhanced activity. Taken together, our study demonstrates proof-of-concept for the application of unbiased mutagenesis screen to identify novel mutations that enhance prime editor performance. Furthermore, we discovered that RT variants (I61R, V101R, S67W) synergize with PEmax and epegRNA to improve prime editing efficiency across edit types, with the strongest improvement observed in introducing small deletions.
CRISPR-Cas systems provide adaptive immunity in prokaryotes, yet how multiple CRISPR-Cas subtypes coexist and coordinate within a single genome remains unclear. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR-Cas3 systems are adjacent to a type I-B system, often sharing a single CRISPR array. Using Thermococcus siculi RG-20 (Tsi) as a model, we show that purified TsiCas6a and TsiCas6b independently recognize and cleave the shared pre-crRNA, producing mature crRNAs with comparable efficiency. Plasmid interference assays further demonstrated that crRNAs produced by either Cas6a or Cas6b enzyme could guide both type I-A and type I-B interference complexes. This interchangeability shows that crRNAs generated by either Cas6a or Cas6b can be loaded into, and function with, both type I-A and type I-B interference complexes. Structural modelling revealed distinct but complementary recognition strategies for Cas6a and Cas6b, and mutational analysis of their RNA-binding residues impaired pre-crRNA cleavage and abolished interference activity. Together, these results uncover a shared-array logic in which Cas6a/Cas6b-compatible processing routes a single pre-crRNA to multiple type I effectors-providing a potential mechanism for subtype co-existence, a plausible explanation for array-less (or "orphan") interference modules, and an evolutionary bet-hedging strategy that prevents Acrs from shutting down immunity wholesale.
Kunming Liu, Chunjiang Ren, Xinyue Liu et al.· Protein & Cell· 0 citations