Blue Light‐Operated CRISPR/Cas13b‐Mediated mRNA Knockdown (Lockdown)

Abstract

that derive from clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques, currently positively reshapes biological studies. [2] Key applications of the specific DNA-recognizing CRISPR/Cas9 systems encompass for example genome editing and transcriptional regulation with high sequence specificity. [2][3][4][5][6] In recent years, these technologies led to major advances in synthetic biology, gene therapy, and gene modification in almost every model organism. Various Cas-variants were subsequently derived from different microorganisms and utilized to overcome some of the limitations restricting in vivo applications, or enabled the recognition of RNA instead of DNA. [2,[7][8][9] Among those, the discovery of the RNA-targeting Cas13 proteins yielded in powerful RNA-editing tools. [9,10] Cas13 belongs to type VI CRISPR effectors and has been utilized for specific knockdown of endogenous RNAs in human cells and manipulation of alternative RNA splicing. [9,11] While the DNAtargeting CRISPR/Cas9 systems require the presence of a short protospacer adjacent motif (PAM) sequence at the editing site, Cas13 is PAM-independent. [9,10] Recent engineering efforts of CRISPR/Cas tools for optogenetic regulation expanded their capabilities including high spatial and temporal control precision, [12][13][14][15][16][17][18][19] however, these systems exclusively target DNA. Only few light-regulated RNA modification tools currently exist, mostly based on RNA interference or short regulatory RNAs, which are CRISPR-independent. [20][21][22][23] We devised here an optogenetic tool to destabilize cellular mRNA by enabling optical control of RNA-targeting CRISPR/ Cas13 systems. For this, we combined a blue light-inducible gene expression switch [24] with the Prevotella sp.-derived Cas13b effector (PspCas13b), [9] resulting in a system termed Lockdown (blue light-operated CRISPR/Cas13b-mediated mRNA knockdown). Blue light activates the gene switch to induce the expression of PspCas13b which, in the presence of a gRNA targeting the mRNA of interest, leads to downregulation of said RNA. Our results demonstrate how Lockdown can be used to regulate cellular processes with blue light through specific mRNA degradation. In our tests, these induced G2 cell cycle arrest and inhibition of cell growth under blue light. [25,26] We further combined Lockdown with the recently published "Blue-OFF" system for synergistic triple-targeted downregulation of proteins. [27,28] To engineer the Lockdown system, we used a split transcription factor based on a modified light-oxygen-voltage domain The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introd...

The Roles of Optogenetics and Technology in Neurobiology: A Review

“…Nowadays, optogenetic can be used to treat eye diseases such as color blindness ( Table 1 and Figure 3 ; Cideciyan et al, 2016 ). The researchers sensitized the cells to light by the expression of light genes that encode light-activated channels or pumps in the remaining retinal cells ( Ostrovsky and Kirpichnikov, 2019 ; Blomeier et al, 2021 ; He et al, 2021 ; Kramer et al, 2021 ; Mickoleit et al, 2021 ). Relevant research has achieved good results in recent years.…”

Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

“…We introduced strongLOV, a mutant El222 transcription factor that requires less light for the same level of activity and thus could reduce phototoxicity. As the El222 optogenetic system is extensively used in organisms other than budding yeast such as mammalian cell lines 119 , bacteria 120 , zebrafish 58 , and plants 121 , the new mutation described here ought to be useful for light control experiments in different fields of biology, as well as contribute to further understanding of LOV-domain proteins photochemistry.…”

Organ-specific COP1 control of BES1 stability adjusts plant growth patterns under shade or warmth

“…24 h before transfection 50,000 cells in 500 ml medium were seeded per well in 24-well plates (Corning). 1,25 mg DNA per well were diluted in 50 ml of OptiMEM and mixed with a PEI/OptiMEM mix [2.5 mL PEI solution (1 mg/ml) in 50 mL OptiMEM] (Blomeier et al, 2021). The ratio between target plasmid (pBES1-pCMVmin::FLUC::tNos) and inducer plasmid (pSV40::PIF4-VP16::pA) was 1:4 (additionally 0,1 mg of normalization plasmid).…”

“…100 ml of transfection mix was added to each well in a drop-wise manner. Medium was exchanged after 4 h. 24 h post transfection luminescence was measured with a Berthold Technologies Centro XS 3 LB 960 Microplate luminometer for FLUC and NanoLuc (normalization) (Blomeier et al, 2021). Cells were lysed with 250 ml lysis buffer and 1mM 1,4-Dithiothreitol (DTT) on ice, and afterwards carefully resuspended as previously described (Baaske et al, 2018).…”