Developing novel CRISPR/CasX editors to generate a CCR5/null immune system

CRISPR/Cas作为编辑导致人类疾病的基因的方法的表征 为创新治疗方法的发展提供了新的机会。七年前，这 细菌免疫系统被改造为切割人类基因组中的特定区域。目前有 超过12项针对癌症、遗传疾病和传染病的临床试验计划或正在进行中， 利用CRISPR编辑。此外，基因编辑可用于切割和潜在切除病毒基因组 这为HIV-1和B型肝炎感染的治疗策略提供了希望， 他人大多数临床前和临床研究使用来源于普通人的Cas9酶。 病原体，以及对Cas9存在体液和细胞免疫的证据可能会阻碍 这种编辑器在患者的子集中，特别是如果使用病毒载体在体内递送，或者如果治疗需要 重复输注（1，2）。 Cas9内切核酸酶家族的这些和其他限制主张开发一种更有效的方法。 各种各样的基因编辑酶的工具箱。最近，几种新的Cas酶被称为CasX（Cas 12 e）和 在来自环境分离株的宏基因组测序数据中鉴定了CasY（Cas 12 d）（3），并且我们 积极开发这些酶用于基因编辑。这些酶的优点包括它们的衍生 从非致病性细菌物种，它们与Cas9相比相对紧凑的大小，以及它们的不同的 原型间隔区邻近基序（PAM）的要求。由指导RNA/CasX核糖核蛋白产生的切割 复合物是不对称的，并产生单链DNA突出端。这种独特的乳沟切割 通过使用位于感兴趣区域侧翼的两个不同的引导RNA来利用以切除靶区域。通过 通过仔细选择引导RNA，我们期望能够在相反的DNA链上产生DNA突出端， 它们在DNA修复过程中退火，基本上切除了中间的 地区此外，我们预期这些不对称突出端也可用于退火成非对称突出端。 外源提供的供体DNA片段，使得切除的区域被新的DNA序列取代。 我们提出的研究将开发CRISPR/CasX，用CCR 5基因取代野生型CCR 5基因。 δ 32等位基因在造血干细胞（HSC）作为下一代方法的发展， HIV-1的治疗应用。已经充分确定，来自对于所述细胞系是纯合的个体的细胞可以被认为是细胞系的细胞。 CCR 5-δ 32等位基因对R5嗜性HIV感染具有抗性，并产生携带两个 这种突变的复制可能会使抗逆转录病毒疗法永久停止。到 为了实现这一目标，我们将开发指导RNA的组合来切除CCR 5基因，并将其替换为 携带突变等位基因的供体DNA下一代测序方法（CIRCLE-seq，MTA-seq） 将用于评估CasX在体外和细胞中的相对效率和特异性。RNA信标 将用于选择其中两个CCR 5等位基因都被替换的细胞。最后，不同比例的野生型 CCR 5-δ 32修饰的HSC将用于人源化免疫缺陷小鼠。数学建模 将定义产生正常数量的免疫细胞所需的编辑的HSC的最小数量 HIV-1挑战后最终，我们的方法将导致新的Cas编辑器的开发，以取代 HIV-1感染所需的细胞基因与那些赋予HIV抗性的基因。

The characterization of CRISPR/Cas as a method to edit genes that contribute to human diseases provides new opportunities for the development of innovative therapeutic approaches. Seven years ago, this bacterial immune system was adapted to cleave specific regions in the human genome. Currently, there are over a dozen clinical trials planned or in process for cancer, genetic disorders and infectious diseases that utilize CRISPR editing. Moreover, gene editing can be used to cleave and potentially excise viral genomes within infected cells, which offers hope for curative strategies for HIV-1 and hepatitis B infections, as well as others. The majority of pre-clinical and clinical studies use the Cas9 enzyme derived from common human pathogens, and evidence for the existence of humoral and cellular immunity to Cas9 could stymie the utility of this editor in a subset of patients, especially if delivered in vivo using viral vectors, or if therapy requires repeated infusions (1, 2). These and other limitations of the family of Cas9 endonucleases argue for the development of a more diverse toolbox of gene editing enzymes. Recently, several novel Cas enzymes termed CasX (Cas12e) and CasY (Cas12d) were identified in metagenomic sequencing data from environmental isolates (3), and we are actively developing these enzymes for gene editing. The advantages of these enzymes include their derivation from non-pathogenic bacterial species, their relatively compact size compared to Cas9, and their distinct protospacer adjacent motif (PAM) requirements. The cut generated by the guide RNA/CasX ribonucleoprotein complex is asymmetrical and generates single stranded DNA overhangs. This unique cleavage cut could then be leveraged to excise a target region by employing two different guide RNAs that flank the area of interest. By careful selection of guide RNAs, we expect that we can generate DNA overhangs on opposite DNA strands that are complementary to each other so they anneal during DNA repair, essentially excising the intervening region. In addition, we expect that these asymmetrical overhangs can also be used to anneal to an exogenously supplied donor DNA fragment, so that the excised region is replaced by new DNA sequence. Our proposed studies will develop CRISPR/CasX to replace the wild-type CCR5 gene with the CCR5- delta 32 allele in hematopoietic stem cells (HSC) as a next generation approach to the development of a therapeutic application for HIV-1. It is well established that cells from individuals who are homozygous for the CCR5-delta 32 allele are resistant to R5-tropic HIV infection, and generating autologous HSCs carrying two copies of this mutation could potentially allow the permanent cessation of anti-retroviral therapy. To accomplish this goal, we will develop combinations of guide RNAs to excise the CCR5 gene, and replace it with donor DNA carrying the mutant allele. Next generation sequencing approaches (CIRCLE-seq, MTA-seq) will be used to assess the relative efficiency and specificity of CasX both in vitro and in cells. An RNA beacon will be used to select for cells in which both CCR5 alleles are replaced. Finally, various proportions of wild-type and CCR5-delta 32-modified HSCs will be used to humanize immunodeficient mice. Mathematical modeling will define the minimum number of edited HSCs necessary to produce normal numbers of immune cells following HIV-1 challenge. Ultimately, our approach will lead to the development of novel Cas editors to replace cellular genes required for HIV-1 infection with those that impart HIV resistance.