CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Time for remodeling: SNF2-family DNA translocases in replication fork metabolism and human disease.

Over the course of DNA replication, DNA lesions, transcriptional intermediates and protein-DNA complexes can impair the progression of replication forks, thus resulting in replication stress. Failure to maintain replication fork integrity in response to replication stress leads to genomic instability and predisposes to the development of cancer and other genetic disorders. Multiple DNA damage and repair pathways have evolved to allow completion of DNA replication following replication stress, thus preserving genomic integrity. One of the processes commonly induced in response to replication stress is fork reversal, which consists in the remodeling of stalled replication forks into four-way DNA junctions. In normal conditions, fork reversal slows down replication fork progression to ensure accurate repair of DNA lesions and facilitates replication fork restart once the DNA lesions have been removed. However, in certain pathological situations, such as the deficiency of DNA repair factors that protect regressed forks from nuclease-mediated degradation, fork reversal can cause genomic instability. In this review, we describe the complex molecular mechanisms regulating fork reversal, with a focus on the role of the SNF2-family fork remodelers SMARCAL1, ZRANB3 and HLTF, and highlight the implications of fork reversal for tumorigenesis and cancer therapy.

MCM8IP activates the MCM8-9 helicase to promote DNA synthesis and homologous recombination upon DNA damage.

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.