RESUMEN

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

RESUMEN

Cohesin is a ring-shaped protein complex that comprises the SMC1, SMC3, and α-kleisin proteins, STAG1/2/3 subunits, and auxiliary factors. Cohesin participates in chromatin remodeling, chromosome segregation, DNA replication, and gene expression regulation during the cell cycle. Mitosis-specific α-kleisin factor RAD21 and meiosis-specific α-kleisin factor REC8 are expressed in embryonic stem cells (ESCs) to maintain pluripotency. Here, we demonstrated that RAD21 and REC8 were involved in maintaining genomic stability and modulating chromatin modification in murine ESCs. When the kleisin subunits were depleted, DNA repair genes were downregulated, thereby reducing cell viability and causing replication protein A (RPA) accumulation. This finding suggested that the repair of exposed single-stranded DNA was inefficient. Furthermore, the depletion of kleisin subunits induced DNA hypermethylation by upregulating DNA methylation proteins. Thus, we proposed that the cohesin complex plays two distinct roles in chromatin remodeling and genomic integrity to ensure the maintenance of pluripotency in ESCs. [BMB Reports 2023; 56(2): 108-113].

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Cromosomas/metabolismo , Meiosis , Mitosis , Cohesinas

RESUMEN

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to singlestranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology. [BMB Reports 2023; 56(2): 102-107].

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Proteína 9 Asociada a CRISPR/genética , Roturas del ADN de Doble Cadena , Genoma

RESUMEN

The coronavirus disease (COVID-19) pandemic has influenced hospital visiting patterns. Although vaccination has decreased infection rates and disease severity, hospital visiting patterns and associated treatment changes related to orthopedics remain unexplored in the Middle East. Therefore, this study aimed to examine the impact of the COVID-19 pandemic and vaccination on individual departments dealing with musculoskeletal disorders in the United Arab Emirates. Relationships between publicly available national data on the number of COVID-19 polymerase chain reaction tests and confirmed and recovered cases during May 2020-July 2021 and hospital data on the number of outpatients, inpatients, operations, and physiotherapy consultations were analyzed. In January 2021, the relationship between vaccination rate and orthopedic unit utilization was evaluated after vaccination campaign initiation. Multifactorial analysis revealed that an increased number of COVID-19-related deaths correlated with a decreased number of joint operations. Negative linear relationships were observed among confirmed and death cases with inpatient treatment and joint operation as well as recovered cases with inpatient treatment. Recovered cases with inpatient treatment and joint operation showed a positive linear relationship. Inpatient spine treatment showed a positive relationship with vaccination rates. The COVID-19 pandemic influenced orthopedic treatment in the Middle East, and vaccination campaigns facilitated inpatient spine treatment.

RESUMEN

Migration of plasma cells (PCs) is crucial for the control of PC survival and antibody production and is controlled by chemokines, most importantly by CXCL12. This study investigated the role of CD99 in CXCL12-induced PC migration. Among B cell subsets in the tonsils, CD99 expression was highest in PCs. CD99 expression increased during in vitro differentiation of germinal center B cells and was highest in PCs. CD99 engagement reduced chemotactic migration of PCs toward CXCL12 and reduced extracellular signal-regulated kinase (ERK) activation by CXCL12. An ERK inhibitor reduced CXCL12-mediated chemotactic migration, which suggests that ERK has a critical role in migration. CD99 engagement did not influence apoptosis, differentiation, or antibody secretion of PCs. We propose a novel role of CD99 in PCs that suppresses ERK activation and chemotactic migration of these cells.

Asunto(s)

Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quimiocina CXCL12/farmacología , Quimiotaxis/efectos de los fármacos , Células Plasmáticas/efectos de los fármacos , Antígeno 12E7 , Animales , Antígenos CD/genética , Linfocitos B/citología , Linfocitos B/metabolismo , Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Centro Germinal/citología , Centro Germinal/metabolismo , Humanos , Inmunohistoquímica , Células L , Ratones , Células Plasmáticas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

ß-agonists are anabolic compounds that promote fat loss and muscle gain, and their administration to livestock may provide economic benefits by increasing growth rate and feed efficiency. For these reasons, ß-agonists are also commonly added to livestock feed as growth promoters. This can introduce a significant risk of secondary human poisoning through intake of contaminated meat. A new method for the simultaneous determination of three ß-agonists (clenbuterol, ractopamine, and zilpaterol) was developed in this study and applied to various meat samples. The limits of quantification, derived through a validation test following Codex guidelines, were 0.2 µg/kg for clenbuterol and zilpaterol, and 0.4 µg/kg for ractopamine. The average recoveries for clenbuterol, ractopamine, and zilpaterol ranged from 109.1% to 118.3%, 95.3% to 109.0%, and 94.1% to 120.0%, respectively. The recovery and coefficient of variation (CV) values fell within the acceptable range according to the Codex guidelines. This method reduced the analysis time without decreasing detection efficiency by modifying the pretreatment steps. This method could be utilized to manage the safety of imported meat products from countries where zilpaterol use is still permitted, thereby improving public health and preventing ß-agonist poisoning due to secondary contamination.