Enhancing CRISPR-mediated homology-directed repair using anti-CRISPR proteins

摘要 遗传性疾病对个人、家庭和社区造成重大损失。其中许多原因 疾病是基因组中的单点突变。尽管在诊断和潜在的遗传学方面取得了进展， 然而，尽管这些疾病的发病基础，但治愈性治疗仍然难以捉摸。CRISPR-Cas9的发现 系统及其编辑人类基因组的能力为治愈性疗法带来了新的希望。然而，在这方面， 纠正基因组中的点突变需要精确修复Cas9诱导的DNA断裂， 同源定向修复（HDR）途径。不幸的是，这种修复途径是低效的，导致低基因组水平。 校正频率Acrigen Biosciences，Inc.正在开创使用抗CRISPR（Acr）蛋白来增强 通过提高HDR的功效进行基因组校正。第一阶段项目将使用最近发现的Acr， 将修复DNA瞬时拴系到Cas9核酸酶，增加修复模板到Cas9核酸酶的局部浓度。 Cas9 DNA切割位点。这将提高HDR的效率和随后对疾病的纠正 导致突变。第一阶段的建议有以下目的：1）建立一个人类细胞报告系统， 评估同源重组的效率。Acrigen将设计CRISPR-Cas9指导RNA和供体 通过HDR将荧光报告基因EGFP转化为BFP的DNA构建体。记者系统将 在人体细胞中得到验证。2)使用Acr技术提高报告人HDR效率。Acrigen将验证结合 Cas9和HDR DNA供体两者的Acr。然后Acrigen将使用Acr将供体DNA瞬时连接到 Cas9，并评估荧光报告系统中同源重组的效率。3)- -- 增强针对SMN 2的HDR。Acrigen将设计Cas9向导RNA和供体DNA模板， 通过编辑SMN 2基因中的单点转换，将截短的SMN蛋白转化为全长SMN。最后，Acrigen 然后将使用Acr来增加脊髓性肌萎缩症（SMA）患者的HDR效率和SMN转换- 衍生细胞在第一阶段结束时，我们将开发出一种新的方法来提高HDR的效率， 细胞，并将验证这种技术，以提高转化SMN作为SMA的潜在治疗。

Abstract Genetic disorders take a significant toll on individuals, families, and communities. The cause of many of these diseases is a single point mutation in the genome. Despite advances in the diagnosis and underlying genetic foundation for these disorders, curative treatments have remained elusive. The discovery of the CRISPR-Cas9 system and its ability to edit human genomes has brought renewed hope for curative therapies. However, correcting point mutations in the genome requires precise repair of the DNA break induced by Cas9 through the homology-directed repair (HDR) pathway. Unfortunately, this repair pathway is inefficient, leading to low genome correction frequencies. Acrigen Biosciences, Inc. is pioneering the use of anti-CRISPR (Acr) proteins to enhance genome correction by increasing the efficacy of HDR. This Phase I project will use a recently discovered Acr to transiently tether repair DNA to the Cas9 nuclease, increasing the local concentration of repair template to the Cas9 DNA cleavage site. This will increase the efficiency of HDR and subsequent correction of the disease causing mutation. The Phase I proposal has the following Aims: 1) Establish a human cell reporter system to assess the efficiency of homologous recombination. Acrigen will design CRISPR-Cas9 guide RNAs and donor DNA constructs to convert the fluorescent reporter EGFP to BFP through HDR. The reporter system will be validated in human cells. 2) Increase reporter HDR efficiency using Acr technology. Acrigen will validate binding of Acr to both Cas9 and the HDR DNA donor. Acrigen will then use Acr to transiently tether the donor DNA to Cas9 and assess the efficiency of homologous recombination in the fluorescent reporter system. 3) Validate Acr- enhanced HDR against SMN2. Acrigen will design Cas9 guide RNAs and donor DNA templates to convert truncated SMN protein to full-length SMN by editing a single point transition in the SMN2 gene. Finally, Acrigen will then use Acr to increase HDR efficiency and SMN conversion in spinal muscular atrophy (SMA) patient- derived cells. At the end of Phase I, we will have developed a new approach to increase HDR efficiency in cells and will have validated this technology to enhance conversion of SMN as a potential cure for SMA.