SBIR Phase I: Development of a safe gene editing system via CRISPR-Cas and Cas inhibitor co-delivery

这个小企业创新研究（SBIR）第一阶段项目的更广泛影响是实现安全有效的人类基因编辑，提供针对无数人类遗传疾病的治疗方法。CRISPR（聚集的、有规律间隔的、短回文重复序列）和CRISPR相关（Cas）基因组成了在不同细菌物种中发现的适应性微生物“免疫系统”，作为抵御病毒感染的防御机制。CRISPR-Cas系统的简单性、可编程性和多功能性使许多生物的遗传修饰成为可能，并为治疗人类疾病提供了巨大的治疗潜力。然而，基于crispr的基因编辑也可能导致脱靶编辑，导致在意外的脱靶编辑中引入突变、插入、缺失或DNA重组。这种影响会导致严重的问题。拟议的技术将开发工具，以安全地将基于crispr的基因编辑转化为体内人类治疗。这项小企业创新研究（SBIR）第一阶段项目旨在推进一项基于病毒编码的CRISPR-Cas抑制剂关闭开关的技术，从而实现对脱靶基因编辑的控制。这些抗CRISPR蛋白是一类新型的强效蛋白抑制剂，可以通过基因编码与CRISPR编辑机制共同递送。该提案将侧重于了解CRISPR编辑动力学，以确定理想的“编辑窗口”，提供最高的治疗效益，同时最大限度地降低脱靶风险。其次，该项目将设计一个系统，用于同时递送CRISPR编辑机制和抗CRISPR抑制剂，以利用这个新的“编辑窗口”。基因调控元件将用于调节系统各组成部分的表达，并为治疗递送系统提供框架。这项工作将在治疗相关的背景下实现安全有效的基因编辑，为翻译提供工具包。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact of this Small Business Innovation Research (SBIR) Phase I project is to enable safe and effective human gene editing, providing therapies targeting myriad human genetic diseases. CRISPR (clustered, regularly interspaced, short palindromic repeats) and CRISPR-associated (Cas) genes compose adaptive microbial `immune systems' found in diverse bacterial species, serving as a defense mechanism against viral infection. The simplicity, programmability, and versatility of CRISPR-Cas systems have enabled genetic modification of many organisms and offer immense therapeutic potential for treating human diseases. However, CRISPR-based gene editing can also cause off-target edits, resulting in the introduction of mutations, insertions, deletions, or DNA restructuring at unintended off-target editing. This effect can cause significant problems. The proposed technology will develop tools to safely translate CRISPR-based gene editing to in vivo human therapeutics. This Small Business Innovation Research (SBIR) Phase I project is to advance a technology based on virus-encoded CRISPR-Cas inhibitor off-switches, enabling control of off-target gene editing. These anti-CRISPR proteins are a novel class of robust protein inhibitors that can be genetically encoded for co-delivery with the CRISPR editing machinery. This proposal will focus on understanding CRISPR editing kinetics to pinpoint the ideal ‘editing window’ providing the highest therapeutic benefit while minimizing off-target risk. Second, the project will design a system for simultaneous delivery of the CRISPR editing machinery along with the anti-CRISPR inhibitor to leverage this new ‘editing window’. Genetic regulatory elements will be used to tune the expression of the various components of the system and provide the framework for a therapeutic delivery system. This work will enable safe and effective gene editing in a therapeutically relevant context, providing a toolkit for translation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.