Dampier Will, Sullivan Neil T, Mell Joshua Chang, Pirrone Vanessa, Ehrlich Garth D, Chung Cheng-Han, Allen Alexander G, DeSimone Mathew, Zhong Wen, Kercher Katherine, Passic Shendra, Williams Jean W, Szep Zsofia, Khalili Kamel, Jacobson Jeffrey M, Nonnemacher Michael R, Wigdahl Brian
1 Department of Microbiology and Immunology, Drexel University College of Medicine , Philadelphia, Pennsylvania.
2 Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine , Philadelphia, Pennsylvania.
AIDS Res Hum Retroviruses. 2018 Nov;34(11):950-960. doi: 10.1089/AID.2017.0274. Epub 2018 Aug 27.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 system has been used to excise the HIV-1 proviral genome from latently infected cells, potentially offering a cure for HIV-infected patients. Recent studies have shown that most published HIV-1 guide RNAs (gRNAs) do not account for the diverse viral quasispecies within or among patients, which continue to diversify with time even in long-term antiretroviral therapy (ART)-suppressed patients. Given this observation, proviral genomes were deep sequenced from 23 HIV-1-infected patients in the Drexel Medicine CNS AIDS Research and Eradication Study cohort at two different visits. Based on the spectrum of integrated proviral DNA polymorphisms observed, three gRNA design strategies were explored: based on the patient's own HIV-1 sequences (personalized), based on consensus sequences from a large sample of patients [broad-spectrum (BS)], or a combination of both approaches. Using a bioinformatic algorithm, the personalized gRNA design was predicted to cut 46 of 48 patient samples at 90% efficiency, whereas the top 4 BS gRNAs (BS4) were predicted to excise provirus from 44 of 48 patient samples with 90% efficiency. Using a mixed design with the top three BS gRNAs plus one personalized gRNA (BS3 + PS1) resulted in predicted excision of provirus from 45 of 48 patient samples with 90% efficiency. In summary, these studies used an algorithmic design strategy to identify potential BS gRNAs to target a spectrum of HIV-1 long teriminal repeat (LTR) quasispecies for use with a small HIV-1-infected population. This approach should advance CRISPR/Cas9 excision technology taking into account the extensive molecular heterogeneity of HIV-1 that persists in situ after prolonged ART.
成簇规律间隔短回文重复序列（CRISPR）相关的Cas9系统已被用于从潜伏感染细胞中切除HIV-1前病毒基因组，这可能为HIV感染患者提供治愈方法。最近的研究表明，大多数已发表的HIV-1引导RNA（gRNA）没有考虑患者体内或患者之间多样的病毒准种，即使在长期接受抗逆转录病毒疗法（ART）抑制的患者中，这些病毒准种也会随着时间不断多样化。基于这一观察结果，在德雷塞尔医学中心艾滋病研究与根除研究队列中，对23名HIV-1感染患者在两次不同就诊时的前病毒基因组进行了深度测序。根据观察到的整合前病毒DNA多态性谱，探索了三种gRNA设计策略：基于患者自身的HIV-1序列（个性化）、基于大量患者样本的共有序列[广谱（BS）]或两种方法的组合。使用生物信息学算法预测，个性化gRNA设计能以90%的效率切割48个患者样本中的46个，而前4个BS gRNA（BS4）预计能以90%的效率从48个患者样本中的44个切除前病毒。使用前三个BS gRNA加一个个性化gRNA的混合设计（BS3 + PS1）预计能以90%的效率从48个患者样本中的45个切除前病毒。总之，这些研究使用了一种算法设计策略来识别潜在的BS gRNA，以靶向一系列HIV-1长末端重复序列（LTR）准种，用于一小部分HIV-1感染人群。考虑到长期ART后原位持续存在的HIV-1广泛分子异质性，这种方法应能推动CRISPR/Cas9切除技术的发展。
