Directed evolution of engineered virus-like particles with improved production and transduction efficiencies.

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of proteins and RNAs, including gene editing agents. We report a system for the laboratory evolution of eVLPs that enables the discovery of eVLP variants with improved properties. The system uses barcoded guide RNAs loaded within DNA-free eVLP-packaged cargos to uniquely label each eVLP variant in a library, enabling the identification of desired variants following selections for desired properties.

Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax.

Efficient prime editing in mouse brain, liver and heart with dual AAVs.

Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%).

Engineered pegRNAs improve prime editing efficiency.

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency.

Radical Ring-Closing/Ring-Opening Cascade Polymerization.

A novel strategy for the synthesis of main-chain polymers through radical ring-closing/ring-opening cascade polymerization is reported. Efficient radical cyclopolymerization was achieved through systematic optimization of the electronic properties of 1,6-diene structures. Fusing 1,6-diene with allylic sulfide or allylic sulfone motifs enabled a ring-closing/ring-opening cascade reaction that provides a strong driving force for the ring-opening polymerization of large macrocyclic monomers.