Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Observing separate spin and charge Fermi seas in a strongly correlated one-dimensional conductor.

An electron is usually considered to have only one form of kinetic energy, but could it have more, for its spin and charge, by exciting other electrons? In one dimension (1D), the physics of interacting electrons is captured well at low energies by the Tomonaga-Luttinger model, yet little has been observed experimentally beyond this linear regime. Here, we report on measurements of many-body modes in 1D gated wires using tunneling spectroscopy. We observe two parabolic dispersions, indicative of separate Fermi seas at high energies, associated with spin and charge excitations, together with the emergence of two additional 1D "replica" modes that strengthen with decreasing wire length. The interaction strength is varied by changing the amount of 1D intersubband screening by more than 45%. Our findings not only demonstrate the existence of spin-charge separation in the whole energy band outside the low-energy limit of the Tomonaga-Luttinger model but also set a constraint on the validity of the newer nonlinear Tomonaga-Luttinger theory.