| Alzheimer’s disease（AD）is the most common form of neurodegenerative disease.The number of individuals suffering from AD are increasing due to the aging population worldwide,leading to significant economic costs and a great burden to the families of AD patients.However,there are no effective interventions to control the aggressive progression of the disease.Previous genetic studies have found over 40 genetic risk genes for AD as well as massive AD-associated single nucleotide polymorphisms（SNPs）,but little is known about the functions of most of them.It is important to develop an in-depth understanding of the roles these genetic risk genes play in AD pathogenesis to gain insights into the invention of effective AD interventions.Previous studies using classical experimental animals,such as the worms,fish,rodents,and monkeys,have provided important knowledge for subsequent,related pathological studies and drug research and development.However,we still do not fully understand the molecular and cellular pathological changes that occur in AD patients.Thus,we urgently need to establish an efficient modeling method to validate the functional genetic risk genes of AD,and develop a human model that can recapitulate the pathological changes in AD.Induced pluripotent stem cells（iPSCs）have been an essential platform for etiology studies of human diseases.While gene-editing technology has been used to introduce mutations into human iPSCs（hiPSCs）to study AD pathogenesis,this approach has limitations such as being time-and labor-consuming and inefficient in selecting positive clones and potential off-targets.Thus,we systemically optimized the procedure of gene-editing,selection,and qualification control in iPSCs.We used electroporation-mediated plasmid delivery for the genome editing,resulting in edited iPSC clones within 2 weeks.Furthermore,the transient expression avoided the side effects induced by long-term Cas9 enzyme overexpression,reducing the off-target risks.Meanwhile,we showed that gene-editing mediated through CRISPR/Cas9 has no effect on the pluripotency and differentiation ability of iPSCs.For example,we used our optimized protocol for iPSC modeling to study the AD risk gene ABCA7,whose function is still unknown to us,in AD pathogenesis.First,we constructed ABCA7 knock-out CRISPR/Cas9 plasmid,and then used transient transfection,puromycin selection and seed-by-seq strategy for for high-throughput selection.Within 2 weeks,we got the positive iPSC clones and found indel mutations in ABCA7.Then,we confirmed that the protein level of ABCA7 was significantly decreased to an undetectable level.Next,we infected the knock-out（KO）clones and healthy wild-type（WT）clones and induced them to differentiate into neurons.We found that the loss-of-function of ABCA7 can cause AD phenotypes,like increased Aβ42 and Aβ42/40 ratio,which are similar to those observed in patients.In addition,we found that the protocadherin family genes and lipid metabolism-related genes were altered in the ABCA7 loss-of-function induced neurons compared to the WT induced neurons in our RNA-sequencing and quantitative PCR data.In summary,we established a quick modeling method to select the functional genetic risk loci of AD as well as human models that can recapitulate the pathological changes in AD,which may help in the study of AD pathology and development of specific AD interventions. |
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