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. Overall design: 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.