Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.