PDF(Pubmed)
Abstract：
Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability. Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential. Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV. Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.
摘要：
简介：随着抗逆转录病毒疗法（ART）的出现，人类免疫缺陷病毒1型（HIV-1）的流行已经减缓。然而,ART不是治愈方法，因此将疾病推向了慢性感染。已经显示出希望的一种潜在的治愈策略是聚集的定期间隔短回文重复(CRISPR)/Cas基因编辑系统。它最近已被证明可以成功地编辑和/或切除感染细胞的整合前病毒，并在体外抑制HIV-1，离体,和体内。这些研究主要使用SpCas9或SaCas9进行。然而,定期发现额外的Cas蛋白，并对这些已知蛋白进行改造。替代的Cas分子对影响HIV-1可能的可靶向区域的前间隔区相邻基序(PAM)具有不同的要求。对Cas蛋白或gRNA柄的其他修饰影响对gRNA和靶标之间的错配的耐受性。在降低脱靶风险的同时,这影响了完全解释HIV-1遗传变异的能力.方法：本手稿致力于使用计算方法检查这些参数选择，以调查Cas编辑器对HIV-1基因编辑的适用性。提名，多样化，狭窄,过滤器（NDNF）管道的安全措施，广泛性，以及任何PAM潜在gRNA库的有效性。该技术用于评估46种不同的Cas编辑的HIV治疗潜力。结果：我们的检查显示，更广泛的PAMs可以提高SaCas9和LbCas12a等编辑的靶向潜力，具有更大的有用gRNA库。而更广泛的PAMs减少了有用的SpCas9gRNA库，但增加了可靶向位置的宽度。对Cas编辑的错配耐受性的研究表明，2-错配耐受性是中靶灵敏度和脱靶特异性之间的理想平衡。在所有Cas编辑中，SpCas-NG和SPRY-Cas9的总体安全数量最高，广泛，和针对HIV的有效gRNA。讨论:目前,更大的蛋白质和更宽的PAMs导致更好的靶向能力。这意味着研究应该针对提供更长的有效载荷或增加当前可用的小型Cas编辑的广度。随着其他Cas编辑的发现和采用，对于HIV-1基因编辑领域的研究人员来说，探索Cas编辑的更广阔世界是很重要的。
