A Strategy Of High Fidelity Cas9 Induced CCR5 And HIV Genome Ablation For Potential AIDS Treatment

AIDS is a public health and social problem of the world.It also is a serious public health issue in China that greatly threatens the health of people.Nowadays,the main treatment for AIDS is highly active antiretroviral therapy(HAART).Although HAART can effectively control HIV,it cannot obliterate the latent HIV-1 provirus and patients may develop drug resistance during the therapy.Therefore,establishing a new treatment is the only hope to completely cure HIV/AIDS.The main target of HIV is activated CD4+ T lymphocytes.Its entry is viainteractions with CD4 and chemokine coreceptors,such as CCR5 or CXCR4.In 2007,an AIDS patient who had developed leukemia received an allogeneic CCR5 gene-deficient hematopoietic stem cell transplantation in German.During 7 years of subsequent follow-up,no symptomatic or laboratorial evidence of HIV infection or AIDS was found.This successful case of AIDS treatment indicates that CCR5 may be an ideal target to cure HIV/AIDS.The development of CRISPR/Cas9 gene editing technology makes it possible to cure many diseases considered incurable before.For example,employing Cas9 system to achieve the ablation of CCR5 receptor in the patient-derived hematopoietic stem cells.The CCR5 deficient stem cell could potentially rebuild a new immune system that wouldn’t been targeted by HIV and provide hope for functional cure of AIDS.Yet,the off-target effect of Cas9 system is the bottleneck of HIV gene therapy that brings us the safety concern.There are currently several strategies to increase the specificity of the Cas9 system,but they also weaken the gene editing efficiency.Thus,our research focused on establishing a system with high fidelity and efficiency to knock out the human CCR5 gene and trying to target latent HIV reservoirs.Method:In order to knock out the human CCR5 gene with high fidelity and efficiency,we designed optimized Cas9 system in different aspect.This system includes Cas9 endonuclease and a single guide RNA.We chose the high specific variant spCas9(eSpCas9 and SpCas9-HF)as the original endonuclease and selected the best sgRNA scaffold.We afterwards designed and screened the best gRNA that knock out CCR5 with high specificity and efficiency.Finally,we used the high fidelity Cas9 to knock out the HIV-1 genome in Jurkat cell.Results:eSpCas9 combined with CCR5-gRNA-2 achieved high fidelity and efficiency in knocking out the human CCR5 gene with a 64.7%knock out rate.The off-target rate in eSpCas9 was lower than WT-Cas9.Using the same CCR5-gRNA-2,SpCas9-HF and WT-Cas9 achieved 60.1%and 35.1%respectively.Moreover,eSpCas9 can knock out latent HIV-1 and obliterate the pro virus.Conclusion:We achieved to knock out the human CCR5 gene and HIV provirus using a CRISPR/Cas9 system with high fidelity and efficiency.This work may provide a potential therapy for the gene therapy for AIDS.