CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Mitosis-specific MRN complex promotes a mitotic signaling cascade to regulate spindle dynamics and chromosome segregation.

The MRE11-RAD50-NBS1 (MRN) complex is well known for participating in DNA damage response pathways in all phases of cell cycle. Here, we show that MRN constitutes a mitosis-specific complex, named mMRN, with a protein, MMAP. MMAP directly interacts with MRE11 and is required for optimal stability of the MRN complex during mitosis. MMAP colocalizes with MRN in mitotic spindles, and MMAP-deficient cells display abnormal spindle dynamics and chromosome segregation similar to MRN-deficient cells. Mechanistically, both MMAP and MRE11 are hyperphosphorylated by the mitotic kinase, PLK1; and the phosphorylation is required for assembly of the mMRN complex. The assembled mMRN complex enables PLK1 to interact with and activate the microtubule depolymerase, KIF2A, leading to spindle turnover and chromosome segregation. Our study identifies a mitosis-specific version of the MRN complex that acts in the PLK1-KIF2A signaling cascade to regulate spindle dynamics and chromosome distribution.