Histone post-translational modifications — cause and consequence of genome function

Prime editing for precise and highly versatile genome manipulation

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

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. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration. 

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

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. 

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

Prime editing (PE) has advantages for small insertion, deletion or point mutations without double-stranded DNA breaks. The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity. Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA, which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs). sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells. We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10120949

Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs

Despite the long-observed correlation between H3K9me3, chromatin architecture and transcriptional repression, how H3K9me3 regulates genome higher-order organization and transcriptional activity in living cells remains unclear. Here we develop EpiGo (Epigenetic perturbation induced Genome organization)-KRAB to introduce H3K9me3 at hundreds of loci spanning megabases on human chromosome 19 and simultaneously track genome organization. EpiGo-KRAB is sufficient to induce de novo heterochromatin-like domain formation, which requires SETDB1, a methyltransferase of H3K9me3. Unexpectedly, EpiGo-KRAB induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac. Ectopic H3K9me3 appears to spread in inactive regions but is largely restricted to transcriptional initiation sites in active regions. Finally, Hi-C analysis showed that EpiGo-KRAB induced to reshape existing compartments. These results reveal the role of H3K9me3 in genome organization could be partially separated from its function in gene repression.

/pdf/simultaneous-epigenetic-perturbation-and-genome-imaging-23efn1pigx.pdf

Simultaneous Epigenetic Perturbation and Genome Imaging Reveal Distinct Roles of H3K9me3 in Chromatin Architecture and Transcription

Abstract The tripartite ParABS system mediates chromosome segregation in a wide range of bacteria. Dimeric ParB was proposed to nucleate on parS sites and spread to neighboring DNA. However, how properly distributed ParB dimers further compact chromosomal DNA into a higher-order nucleoprotein complex for partitioning remains poorly understood. Here, using a single-molecule approach, we show that tens of Bacillus subtilis ParB (Spo0J) proteins can stochastically multimerize on and stably bind to nonspecific DNA. The introduction of CTP promotes the formation and diffusion of the multimeric ParB along DNA, offering an opportunity for ParB proteins to further forgather and cluster. Intriguingly, ParB multimers can recognize parS motifs and are more inclined to remain immobile on them. Importantly, the ParB multimer features distinct capabilities of not only bridging two independent DNA molecules but also mediating their transportation, both of which are enhanced by the presence of either CTP or parS in the DNA. These findings shed new light on ParB dynamics in self-multimerization and DNA organization and help to better comprehend the assembly of the ParB-DNA partition complex.

/pdf/stochastically-multimerized-parb-orchestrates-dna-assembly-28zwbxy8.pdf

Stochastically multimerized ParB orchestrates DNA assembly as unveiled by single-molecule analysis

The local density of H3K9me3 dictates the stability of HP1α condensates-mediated genomic interactions.

Abstract The dynamics of genomic loci pairs and their interactions are essential for transcriptional regulation and genome organization. However, a robust method for tracking pairwise genomic loci in living cells is lacking. Here we developed a multicolor DNA labeling system, mParSpot (multicolor ParSpot), to track pairs of genomic loci and their interactions in living cells. The mParSpot system is derived from the ParB/ParS in the parABS system and Noc/NBS in its paralogous nucleoid occlusion system. The insertion of 16 base-pair palindromic ParSs or NBSs into the genomic locus allows the cognate binding protein ParB or Noc to spread kilobases of DNA around ParSs or NBSs for loci-specific visualization. We tracked two loci with a genomic distance of 53 kilobases and measured their spatial distance over time. Using the mParSpot system, we labeled the promoter and terminator of the MSI2 gene span 423 kb and measured their spatial distance. We also tracked the promoter and terminator dynamics of the MUC4 gene in living cells. In sum, the mParSpot is a robust and sensitive DNA labeling system for tracking genomic interactions in space and time under physiological or pathological contexts.

Tracking pairwise genomic loci by the ParB–ParS and Noc-NBS systems in living cells

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