Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer.

Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9-DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151-1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.

Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation.

Staphylococcus aureus Cas9 (SaCas9) is an RNA-guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single-molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6- and 18-bp of PAM-proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA-bound SaCas9 a persistent block to DNA-tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM-distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.

YTHDF1 Regulates Pulmonary Hypertension through Translational Control of MAGED1.

Rationale: Posttranscriptional modifications are implicated in vascular remodeling of pulmonary hypertension (PH). m6A (N6-methyladenosine) is an abundant RNA modification that is involved in various biological processes. Whether m6A RNA modification and m6A effector proteins play a role in pulmonary vascular remodeling and PH has not been demonstrated.Objectives: To determine whether m6A modification and m6A effectors contribute to the pathogenesis of PH.Methods: m6A modification and YTHDF1 expression were measured in human and experimental PH samples. RNA immunoprecipitation analysis and m6A sequencing were employed to screen m6A-marked transcripts. Genetic approaches were employed to assess the respective roles of YTHDF1 and MAGED1 in PH. Primary cell isolation and cultivation were used for function analysis of pulmonary artery smooth muscle cells (PASMCs).Measurements and Main Results: Elevated m6A levels and increased YTHDF1 protein expression were found in human and rodent PH samples as well as in hypoxic PASMCs. The deletion of YTHDF1 ameliorated PASMC proliferation, phenotype switch, and PH development both in vivo and in vitro. m6A RNA immunoprecipitation analysis identified MAGED1 as an m6A-regulated gene in PH, and genetic ablation of MAGED1 improved vascular remodeling and hemodynamic parameters in SU5416/hypoxia mice. YTHDF1 recognized and promoted translation of MAGED1 in an m6A-dependent manner that was absent in METTL3-deficient PASMCs. In addition, MAGED1 silencing inhibited hypoxia-induced proliferation of PASMCs through downregulating PCNA.Conclusions: YTHDF1 promotes PASMC proliferation and PH by enhancing MAGED1 translation. This study identifies the m6A RNA modification as a novel mediator of pathological changes in PASMCs and PH.

m6 A reader protein YTHDF2 regulates spermatogenesis by timely clearance of phase-specific transcripts.

OBJECTIVES: Accumulating evidences show that the regulatory network of m6 A modification is essential for mammalian spermatogenesis. However, as an m6 A reader, the roles of YTHDF2 remain enigmatic due to the lack of a proper model. Here, we employed the germ cell conditional knockout mouse model and explored the function of YTHDF2 in spermatogenesis. MATERIALS AND METHODS: Ythdf2 germ cell conditional knockout mice were obtained by crossing Ythdf2-floxed mice with Vasa-Cre and Stra8-Cre mice. Haematoxylin and eosin (HE) staining, immunofluorescent staining and Western blotting were used for phenotyping. CASA, IVF and ICSI were applied for sperm function analysis. RNA-seq, YTHDF2-RIP-seq and quantitative real-time PCR were used to explore transcriptome changes and molecular mechanism analysis. RESULTS: Our results showed that YTHDF2 was highly expressed in spermatogenic cells. The germ cell conditional knockout males were sterile, and their sperm displayed malformation, impaired motility, and lost fertilization ability. During differentiated spermatogonia transiting to pachytene spermatocyte, most m6 A-modified YTHDF2 targets that were degraded in control germ cells persisted in pachytene spermatocytes of Ythdf2-vKO mice. These delayed mRNAs were mainly enriched in pathways related to the regulation of transcription, and disturbed the transcriptome of round spermatid and elongated spermatid subsequently. CONCLUSION: Our data demonstrate that YTHDF2 facilitates the timely turnover of phase-specific transcripts to ensure the proper progression of spermatogenesis, which highlights a critical role of YTHDF2 in spermatogenesis.