While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types. ll

Angelman Syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited copy of UBE3A, an imprinted gene expressed biallelically in most tissues, but expressed exclusively from the maternal allele in neurons. Active transcription of the neuron-specific long non-coding RNA (lncRNA), UBE3A-ATS, has been shown to silence paternal UBE3A. We hypothesized that alternative splicing factors RBFOX2 and RBFOX1 might mediate splicing changes and result in the transcription of UBE3A-ATS in neurons. We found that RBFOX2 and RBFOX1 both bind to UBE3A-ATS transcript in neurons, but are not required for gene expression and/or neuron-specific processing in the SNURF/SNRPN-UBE3A region. However, we found that depletion of RBFOX2 causes a proliferation phenotype in immature neural cultures, suggesting that RBFOX2 is involved in division versus differentiation decisions in iPSC-derived neural progenitors. Absence of RBFOX2 also altered the expression of some genes that are important for glutamatergic neocortical development and Wnt-Frizzled signalling in mature neuronal cultures. Our data show that while RBFOX1 and RBFOX2 do not mediate neuron-specific processing of UBE3A-ATS, these proteins play important roles in developing neurons and are not completely functionally redundant.

Medication non-adherence is a concern in chronic disease management. Currently, there is no scale that characterizes sufficient non-adherent reasons for practical use in the Chinese population. This study developed and validated the Chinese version of the Medication Adherence Reasons Scale (ChMAR-Scale) and described non-adherence reasons in adult patients taking blood pressure medicine in Taiwan. A forward–backward procedure was used to translate the original MAR-Scale, and new items pertinent to cultural differences were added. Patients aged above 20 years old who were taking blood pressure medicine were recruited from a regional hospital and eight community pharmacies in the Taipei metropolitan area. Data analyses were conducted with IBM SPSS 19 (Armonk, NY, USA). Exploratory factor analysis revealed six domains, including belief, self-perception, forgetfulness, management, availability, and miscellaneous issues, with Cronbach’s alphas ranging from 0.649 to 0.852, item-total correlations ranging from 0.362 to 0.719, and factor loadings ranging from 0.365 to 0.775. Criterion-related validity with the visual analog scale and two global items were 0.525, 0.436, and 0.502. Forgetfulness, belief issues, and self-perception issues were the most common non-adherence reasons. In conclusion, the ChMAR-Scale showed good psychometric properties and identified more reasons for medication non-adherence than other existing scales. Healthcare providers should be vigilant of these problems while consulting patients.