Proposed field: inherited retinal dystrophies and gene therapy Title of project: Developing CRISPR gRNA delivery strategies for the treatment of retin

CRISPR系统的发现彻底改变了我们操纵基因组的能力。CRISPR-Cas构建体需要指导RNA（gRNA）来指导编辑靶序列，其可以通过AAV递送。然而，AAV的使用带来了限制，包括包装限制、长期Cas过表达和成本效益，这是由于每个靶标需要独特的AAV。玻璃体内递送的化学修饰的RNA反义寡核苷酸已经显示出在光感受器中重定向剪接。如果Cas-gRNA可以被类似地修饰和递送，这将允许我们独立地递送多个gRNA，允许多个靶标的同时或交错沉默，并应用于研究和治疗。本DPhil的目的是研究通过玻璃体内途径将化学修饰的gRNA单独递送至视网膜，并检查这些gRNA的剂量、时机和修饰对其功效的影响。为此，该项目的关键目标是：1）体外敲除EGFP：a.在HEK 293 TdEGFP细胞中筛选靶向EGFP的gRNA B. HEK 293 TdEGFP细胞中的无测试修饰的（在3'和5'端的3个核苷酸处具有2 '0-甲基和硫代磷酸酯键）、未修饰的EGFP靶向和乱序gRNA。选择3'和5'修饰，因为它们具有高编辑效力、过度修饰的潜在毒性和广泛的商业可得性。C. 2）gRNA行为、递送和功效的体内研究a.表征SpCas 9 EGFP和dSpCas 9 KRA B/Nrl.EGFP小鼠B。将游离的修饰的和未修饰的靶向EGFP的和乱序的gRNA玻璃体内递送至spCas 9小鼠。C.使用OCT成像研究视网膜炎症和厚度d的毒性。通过qPCR e检查gRNA在视网膜层中的渗透。使用TIDE、体内荧光、IHC、蛋白质印迹和qPCR检查EGFP敲低的影响在HEK 293 T细胞系中筛选靶向SaCas 9和spCas 9的gRNA B.按照上述步骤1，在HEK 293 TdEGFP细胞中测试化学修饰和未修饰的gRNA。C.用TIDE和蛋白质印迹研究敲低的功效。4)开发靶向EGFP的gRNA以用于SaCas 9。a.按照上述步骤1进行操作。其目的是使用该指南与在GRK 1启动子下表达SaCas 9的SaCas 9小鼠一起使用，与Nrl.EGFP小鼠系杂交，以创建在光感受器中具有稳健SaCas 9和EGFP表达的小鼠，以确保化学修饰的gRNA与SaCas 9具有同等活性。提出该步骤也是因为SpCas 9小鼠系的表征显示出在感光层中出乎意料地差的SpCas 9表达，因此需要具有更稳健的感光器Cas9表达的小鼠。5)靶向EGFP的Sa gRNA的体内研究a. 6）自靶向gRNA行为的体内研究a.在两种Cas9小鼠系中，研究递送自靶向gRNA以沉默Cas9表达同时维持初始良好EGFP敲低的时机和剂量。

The discovery of CRISPR systems has revolutionised our ability to manipulate the genome. CRISPR-Cas constructs require a guide RNA (gRNA) to direct editing to a target sequence, which can be delivered via an AAV. However, the use of AAVs come with constraints, including packaging limitations, long-term Cas over-expression, and cost-effectiveness due to the need for a unique AAV for each target. Intravitreally delivered chemically modified RNA antisense oligonucleotides have been shown to redirect splicing in photoreceptors. If a Cas-gRNA can be similarly modified and delivered, this would allow us to deliver multiple gRNAs independently, allowing simultaneous or staggered silencing of multiple targets, with applications in research and therapy. The aim of this DPhil is to investigate the separate delivery of chemically modified gRNAs to the retina by an intravitreal route and to examine the impact of the dosage, timing, and modification of these gRNAs on their efficacy. To this end, the key goals of the project are: 1) In vitro knockdown of EGFP: a. Screen gRNAs targeting EGFP in HEK293TdEGFP cells b. Test free modified (with 2'O-methyl and phosphorothioated bonds at the 3 nucleotides at the 3' and 5' ends), unmodified EGFP-targeting and scrambled gRNA in HEK293TdEGFP cells. 3' and 5' modifications were chosen given their high editing efficacy, potential toxicity of excessive modification, and wide commercial availability. c. Investigate efficacy of knockdown with TIDE, fluorescence, western blot, and qPCR 2) In vivo investigation of gRNA behaviour, delivery, and efficacy a. Characterise the SpCas9EGFP and dSpCas9KRAB/Nrl.EGFP mouse b. Deliver free modified and unmodified EGFP-targeting and scrambled gRNA intravitreally to the spCas9 mouse. c. Investigate toxicity using OCT imaging for retinal inflammation and thickness d. Examine penetration of the gRNA in the retinal layers by qPCR e. Examine effects of EGFP knockdown using TIDE, in vivo fluorescence, IHC, Western blot and qPCR 3) Development of self-targeting SaCas9 and spCas9 gRNAs a. Screening of gRNAs targeting SaCas9 and spCas9 in a HEK293T cell line b. As per step 1 above, testing chemically modified and unmodified gRNAs in HEK293TdEGFP cells. c. Investigate efficacy of knockdown with TIDE and western blot. 4) Development of a gRNA targeting EGFP to be used with SaCas9. a. Steps as per step 1 above. The aim of this is to use this guide with an SaCas9 mouse expressing SaCas9 under a GRK1 promoter, crossed with an Nrl.EGFP mouse line to create a mouse with robust SaCas9 and EGFP expression in photoreceptors, to ensure that chemically modified gRNAs are equally active with SaCas9. This step is also proposed because characterisation of the SpCas9 mouse lines showed unexpectedly poor SpCas9 expression in the photoreceptor layer, thus a mouse with more robust photoreceptor Cas9 expression was required. 5) In vivo investigation of Sa gRNA targeting EGFP a. As per step 2 above but using the SaCas9 mouse line 6) In vivo investigation of self-targeting gRNA behaviour a. In both Cas9 mouse lines, investigating the timing and dosage of delivery of a self-targeting gRNA to silence Cas9 expression while maintaining initial good EGFP knockdown.