RESUMEN
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 (SaCas9) mRNA in each lentivirus-like bionanoparticle (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9. Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.
Asunto(s)
Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Nanopartículas/metabolismo , Transfección/métodos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Nanopartículas/química , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Staphylococcus aureus/química , Staphylococcus aureus/enzimologíaRESUMEN
Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.
Asunto(s)
Sistemas CRISPR-Cas , Conversión Génica , Edición Génica , Células HEK293 , HumanosRESUMEN
BACKGROUND: Obesity is a multifactorial metabolic disease resulting from behavioral and genetic factors. Obesity is linked to diabetes mellitus and hypertension, which are considered as major risk factors for chronic kidney disease (CKD); moreover, it has a direct effect on developing CKD and end stage renal disease (ESRD). Here was aimed to examine the association between uncoupling protein 2 (UCP2) gene expression and obesity in CKD patients. METHODS: UCP2 gene expression was analyzed by real time polymerase chain reaction (RT-PCR) in 93 participants divided into three groups. The groups included 31 non-obese CKD patients, 31 obese CKD patients, and 31 healthy, age-matched, unrelated volunteers as a control group. RESULTS: UCP2 gene expression was significantly relevant when comparing the non-obese CKD and obese CKD groups to the control group (p< 0.001). No significant association was found when the groups were compared by gender; Chi-square (X2) was 2.38 and p= 0.304. A significant negative correlation was found between UCP2 gene expression and BMI in CKD (p< 0.05). CONCLUSION: These results indicate that UCP2 gene expression plays a significant role as a risk factor for obesity in CKD patients.