Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Mapping the Genetic Interaction Network of PARP inhibitor Response.

Genetic interactions have long informed our understanding of the coordinated proteins and pathways that respond to DNA damage in mammalian cells, but systematic interrogation of the genetic network underlying that system has yet to be achieved. Towards this goal, we measured 147,153 pairwise interactions among genes implicated in PARP inhibitor (PARPi) response. Evaluating genetic interactions at this scale, with and without exposure to PARPi, revealed hierarchical organization of the pathways and complexes that maintain genome stability during normal growth and defined changes that occur upon accumulation of DNA lesions due to cytotoxic doses of PARPi. We uncovered unexpected relationships among DNA repair genes, including context-specific buffering interactions between the minimally characterized AUNIP and BRCA1-A complex genes. Our work thus establishes a foundation for mapping differential genetic interactions in mammalian cells and provides a comprehensive resource for future studies of DNA repair and PARP inhibitors.

A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.