Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo

There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. The flexibility of CRISPR, coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool (BLU-VIPR), that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo. To achieve optogenetic base editing of an endogenous gene, 4x10^4 293FT cells were seeded in black walled, optical bottom 96 well plates and transfected and transfected with 50 ng pRS0035 and 50 ng BLU-VIPR plasmids with NEAT1 targeting or non-targeting gRNA. Twenty-four hours post-transfection, cells were exposed to pulses (20 seconds ON, 60 seconds OFF) of 1 mW/cm^2 of 470 nm light for 48 hours or kept in the dark. Light-exposed cells were harvested and sorted for mCherry expression on a Sony SH800 cell sorter (Sony Biotechnology). Genomic DNA was extracted from cells exposed to light, and from cells kept in the dark, using the Monarch Genomic DNA Purification Kit following the manufacturer’s protocol (New England Biolabs). The targeted region in NEAT1 was amplified by genomic PCR. Amplicons were first sequenced by Sanger sequencing and analyzed with EditR software21. Next, to obtain precise quantification of edits, we performed Illumina sequencing. Briefly, a sequencing library was generated by equimolar pooling of indexed genomic PCR products of endogenous NEAT1. The PCR products were isolated using QIAquick Gel Extraction Kit (Qiagen) and quality control was performed with the Agilent High Sensitivity D1000 ScreenTape System (Agilent Technologies). The pooled library was sequenced using the MiSeq System with the MiSeq Micro Flow Cell (4 million reads) paired-end i7 indexed reads (150 cycles). Fastq files were demultiplexed and analyzed with CRISPResso2 for base editing.

目前尚缺乏能够同时实现精准且时空可控的基因扰动工具。CRISPR（成簇规律间隔短回文重复序列）的灵活性，结合可控光源所能提供的无与伦比的时空分辨率，使得光遗传CRISPR（optogenetic CRISPR）成为精准时空基因扰动的理想解决方案。本研究报道了一款新型光遗传CRISPR工具BLU-VIPR，其不同于主流的拆分型Cas（split-Cas）设计策略，转而聚焦于向导RNA（gRNA）生成的光遗传调控。本研究围绕一种新型高效蓝光激活转录因子及核酶侧翼向导RNA（gRNA）构建了BLU-VIPR系统。BLU-VIPR为遗传编码型设计，可实现从单条mRNA转录本中精准切出多条向导RNA，简化了时空基因扰动流程，并支持多种类型的光遗传CRISPR应用，包括插入缺失突变（indels）、CRISPR激活系统（CRISPRa）以及碱基编辑（base editing）。BLU-VIPR同样可在体内应用于此前难以实现光遗传基因编辑的细胞，并成功在体内T淋巴细胞中完成光遗传基因编辑。为实现内源基因的光遗传碱基编辑，我们将4×10^4个293FT细胞接种于黑色壁、光学底部的96孔板中，随后用50 ng pRS0035质粒与50 ng靶向或非靶向NEAT1的向导RNA（gRNA）关联BLU-VIPR质粒进行转染。转染24小时后，将细胞置于强度为1 mW/cm²、波长470 nm的脉冲光（亮20秒、暗60秒）下照射48小时，或置于黑暗环境中培养。将光照组细胞收集后，通过索尼SH800细胞分选仪（Sony Biotechnology）基于mCherry表达进行分选。采用Monarch基因组DNA纯化试剂盒（New England Biolabs），按照制造商说明书分别从光照组与黑暗对照组细胞中提取基因组DNA。通过基因组PCR扩增NEAT1的靶向区域，首先采用桑格测序法对扩增产物进行测序，并通过EditR软件21进行数据分析。随后，为精准定量编辑事件，我们开展了Illumina测序。简言之，我们将内源NEAT1的带索引基因组PCR产物以等摩尔浓度混合，构建测序文库。PCR产物采用QIAquick凝胶回收试剂盒（Qiagen）进行纯化，并通过安捷伦高灵敏度D1000 ScreenTape系统（Agilent Technologies）完成质量控制。混合后的文库采用MiSeq系统搭配MiSeq微流动池（400万条读长）进行双端i7索引测序（150个循环）。对Fastq文件进行解多重索引后，通过CRISPResso2软件分析碱基编辑结果。