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。其中许多问题的原因 疾病是基因组中的单点突变。尽管在诊断和潜在的基因方面取得了进展 在这些疾病的基础上，根治方法仍然难以捉摸。CRISPR-CAS9的发现 该系统及其编辑人类基因组的能力为根治疗法带来了新的希望。然而， 纠正基因组中的点突变需要精确修复Cas9通过 同源定向修复(HDR)途径。不幸的是，这种修复途径效率低下，导致基因组低。 校正频率。Acrigen Biosciences，Inc.率先使用抗CRISPR(ACR)蛋白来增强 通过增加HDR的效率来进行基因组校正。该第一阶段项目将使用最近发现的ACR来 将修复DNA瞬时拴在Cas9核酸酶上，将修复模板的局部浓度增加到 Cas9DNA裂解位点。这将提高HDR的效率和随后对疾病的纠正 导致突变。第一阶段建议的目标如下：1)建立人体细胞报告系统，以 评估同源重组的效率。Acrigen将设计CRISPR-Cas9指南RNA和供体 构建DNA，通过HDR将荧光报告基因EGFP转化为BFP。记者制度将是 在人类细胞中得到了验证。2)利用ACR技术提高报告器HDR效率。Acrigen将验证绑定 对CAS9和HDR DNA供体的ACR。然后，Acrigen将使用ACR将捐赠者的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)验证ACR- 针对SMN2增强了HDR。Acrigen将设计Cas9指南RNA和供体DNA模板以进行转换 通过编辑SMN基因中的单点转换，将SMN蛋白截短为全长SMN。最后，Acrigen 然后将使用ACR来提高脊髓性肌萎缩症(SMA)患者的HDR效率和SMN转化率- 派生的细胞。在第一阶段结束时，我们将开发一种新的方法来提高HDR效率 细胞，并将验证这项技术，以提高SMN的转化，作为一种潜在的SMA治愈。

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.