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.

A prime editor mouse to model a broad spectrum of somatic mutations in vivo.

Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline.

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%).

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBB) to wild type (HBB) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBB levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors.

A molecular glue approach to control the half-life of CRISPR-based technologies.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life.

Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors.

The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV approaches based on trans-splicing inteins have been used. Here we show that, compared with dual-AAV systems, AAVs with size-optimized genomes incorporating compact adenine base editors (ABEs) enable efficient editing in mice at similar or lower doses.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types.

Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins.

Search-and-replace genome editing without double-strand breaks or donor DNA.

Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates.

Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin.

Thrombin normally cleaves fibrinogen to promote coagulation; however, binding of thrombomodulin to thrombin switches the specificity of thrombin toward protein C, triggering the anticoagulation pathway. The W215A thrombin mutant was reported to have decreased activity toward fibrinogen without significant loss of activity toward protein C. To understand how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium exchange experiments (HDXMS), accelerated molecular dynamics (AMD) simulations, and activity assays were carried out to compare the dynamics of Trp215 mutants with those of wild type (WT) thrombin.