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构建需要一个引导RNA （gRNA）来指导编辑到目标序列，这可以通过AAV传递。然而，AAV的使用有一些限制，包括包装限制，长期的Cas过表达，以及由于每个靶标需要独特的AAV而导致的成本效益。玻璃体内传递化学修饰的RNA反义寡核苷酸已被证明可以改变光感受器剪接的方向。如果一个Cas-gRNA可以类似地修饰和递送，这将使我们能够独立递送多个grna，允许同时或交错沉默多个靶标，在研究和治疗中应用。本DPhil的目的是研究通过玻璃体内途径将化学修饰的grna单独递送到视网膜，并检查这些grna的剂量、时间和修饰对其疗效的影响。为此，本项目的主要目标是：1)体外敲除EGFP: a.筛选HEK293TdEGFP细胞中靶向EGFP的gRNA； b.在HEK293TdEGFP细胞中检测游离修饰（3‘端和5’端3个核苷酸具有2' o -甲基和磷酸化键）、未修饰的靶向EGFP和乱序gRNA。选择3‘和5’修饰是考虑到它们的高编辑功效、过度修饰的潜在毒性和广泛的商业可用性。c.利用TIDE、荧光、western blot和qPCR研究敲除的效果2)gRNA行为、传递和功效的体内研究a.表征SpCas9EGFP和dSpCas9KRAB/Nrl。b.将经修饰的和未修饰的靶向EGFP和打乱的gRNA通过体外注入spCas9小鼠。c.利用OCT成像研究视网膜炎症和厚度的毒性d.通过qPCR检测gRNA在视网膜层中的渗透e.使用TIDE、体内荧光、免疫组化、Western blot和qPCR检测EGFP敲低的影响e.使用自靶向SaCas9和spCas9 gRNA的开发a.在HEK293T细胞系中筛选靶向SaCas9和spCas9的gRNA b.按照上述第1步，在HEK293TdEGFP细胞中检测化学修饰和未修饰的gRNA。c.用TIDE和western blot观察敲低的效果。4)开发靶向EGFP的gRNA用于SaCas9。a.按照上述步骤1的步骤。目的是将该指南应用于在GRK1启动子下表达SaCas9的SaCas9小鼠，并与Nrl杂交。EGFP小鼠细胞系，以创建具有强大的SaCas9和EGFP光感受器表达的小鼠，以确保化学修饰的grna与SaCas9具有相同的活性。之所以提出这一步骤，还因为SpCas9小鼠系的表征显示，SpCas9在光感受器层的表达出乎意料地差，因此需要具有更强的光感受器Cas9表达的小鼠。5) Sa gRNA靶向EGFP的体内研究a.根据上述步骤2，使用SaCas9小鼠系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的时间和剂量，以沉默Cas9表达，同时保持初始良好的EGFP敲除。

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.