Target binding and residence:a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms,and holds tremendous promise in clinical applications.The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA(sgRNA)at a given site.However,little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks(DSBs).We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target,contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing.Here,we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing,and the opportunity this presents to optimize Cas9-based technology.

A green repair pathway for spent spinel cathode material:Coupled mechanochemistry and solid-phase reactions

A way of directly repairing spent lithium-ion battery cathode materials is needed in response to environmental pollution and resource depletion.In this work,we report a green repair method involving coupled mechano-chemistry and solid-state reactions for spent lithium-ion batteries.During the ball-milling repair process,an added manganese source enters into the degraded LiMn_(2)O_(4)(LMO)crystal structure in order to fill the Mn vacancies formed by Mn deficiency due to the Jahn–Teller effect,thereby repairing the LMO's chemical composition.An added carbon source acts not only as a lubricant but also as a conductor to improve the material's electrical conductivity.Meanwhile,mechanical force reduces the crystal size of the LMO particles,increasing the amount of active sites for electrochemical reactions.Jahn–Teller distortion is successfully suppressed by cation disorder in the LMO material.The cycling stability and rate performance of the repaired cathode material are thereby greatly improved,with the discharge specific capacity being more than twice that of commercial LMO.The proposed solid-state mechanochemical in situ repair process,which is safe for the environment and simple to use,may be extended to the repair of other waste materials without consuming highly acidic or alkaline chemical reagents.

Two types of auditory glutamatergic synapses and their implications for repairing damaged central auditory pathways

For the mammalian brain to process and decipher the rich panoply of sounds that abound in the world, nature has evolved an elegant collection of neural circuits dedicated to this task. Indeed, the complexity, variety and number of neural pathways devoted to computing auditory information is unique among sensory modalities （Kaas, 2008）. After the initial sensorineural encoding of sound at the level of the cochlea, auditory information is processed in several lower brainstem centers and eventually converges in the midbrain, at the level of the inferior colliculus （Wenstrup, 2005）, Subsequently, auditory information is transferred through the thalamus, the medial geniculate body, and then the auditory cortex （Winer et al., 2005; Razak and Fuzessery, 2010; Hackett, 2011; Lee and Sherman, 2011; Lee and Winer, 2011;

Establishing the homologous recombination score threshold in metastatic prostate cancer patients to predict the efficacy of PARP inhibitors

Background:The homologous recombination deficiency(HRD)score serves as a promising biomarker to iden-tify patients who are eligible for treatment with PARP inhibitors(PARPi).Previous studies have suggested a 3-biomarker Genomic Instability Score(GIS)threshold of≥42 as a valid biomarker to predict response to PARPi in patients with ovarian cancer and breast cancer.However,the GIS threshold for prostate cancer(PCa)is still lacking.Here,we conducted an exploratory analysis to investigate an appropriate HRD score threshold and to evaluate its ability to predict response to PARPi in PCa patients.Methods:A total of 181 patients with metastatic castration-resistant PCa were included in this study.Tumor tissue specimens were collected for targeted next-generation sequencing for homologous recombination repair(HRR)genes and copy number variation(CNV)analysis.The HRD score was calculated based on over 50,000 single-nucleotide polymorphisms(SNP)distributed across the human genome,incorporating three SNP-based as-says:loss of heterozygosity,telomeric allelic imbalance,and large-scale state transition.The HRD score threshold was set at the last 5th percentile of the HRD scores in our cohort of known HRR-deficient tumors.The relation-ship between the HRD score and the efficacy in 16 patients of our cohort who received PARPi treatment were retrospectively analyzed.Results:Genomic testing was succeeded in 162 patients.In our cohort,61 patients(37.7%)had HRR mutations(HRRm).BRCA mutations occurred in 15 patients(9.3%).The median HRD score was 4(ranged from 0 to 57)in the total cohort,which is much lower than that in breast and ovarian cancers.Patients who harbored HRRm and BRCA or TP53 mutations had higher HRD scores.CNV occured more frequently in patients with HRRm.The last 5th percentile of HRD scores was 43 in the HRR-mutant cohort and consequently HRD high was defined as HRD scores≥43.In the 16 patients who received PARPi in our cohort,4 patients with a high HRD score achieved an objective response rate(ORR)of 100%while 12 patients with a low HRD score achieved an ORR of 8.3%.Progression-free survival(PFS)in HRD high patients was longer compared to HRD low patients,regardless of HRRm.Conclusions:A HRD score threshold of 43 was established and preliminarily validated to predict the efficacy of PARPi in this study.Future studies are needed to further verify this threshold.

Genetic signatures of ERCC1 and ERCC2 expression,along with SNPs variants,unveil favorable prognosis in SCLC patients undergoing platinum-based chemotherapy

Background:Platinum chemotherapy(CT)remains the backbone of systemic therapy for patients with smallcell lung cancer(SCLC).The nucleotide excision repair(NER)pathway plays a central role in the repair of the DNA damage exerted by platinum agents.Alteration in this repair mechanism may affect patients’survival.Materials and Methods:We conducted a retrospective analysis of data from 38 patients with extensive disease(ED)-SCLC who underwent platinum-CT at the Clinical Oncology Unit,Careggi University Hospital,Florence(Italy),from 2015 to 2020.mRNA expression analysis and single nucleotide polymorphism(SNP)characterization of three NER pathway genes—namely ERCC1,ERCC2,and ERCC5—were performed on patient tumor samples.Results:Overall,elevated expression of ERCC genes was observed in SCLC patients compared to healthy controls.Patients with low ERCC1 and ERCC5 expression levels exhibited a better median progression-free survival(mPFS=7.1 vs.4.9 months,p=0.39 for ERCC1 and mPFS=6.9 vs.4.8 months,p=0.093 for ERCC5)and overall survival(mOS=8.7 vs.6.0 months,p=0.4 for ERCC1 and mOS=7.2 vs.6.2 months,p=0.13 for ERCC5).Genotyping analysis of five SNPs of ERCC genes showed a longer survival in patients harboring the wild-type genotype or the heterozygous variant of the ERCC1 rs11615 SNP(p=0.24 for PFS and p=0.14 for OS)and of the rs13181 and rs1799793 ERCC2 SNPs(p=0.43 and p=0.26 for PFS and p=0.21 and p=0.16 for OS,respectively)compared to patients with homozygous mutant genotypes.Conclusions:The comprehensive analysis of ERCC gene expression and SNP variants appears to identify patients who derive greater survival benefits from platinum-CT.