Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

Knapp, David J.H.F.; Michaels, Yale S.; Jamilly, Max; Ferry, Quentin R. V.; Barbosa, Hector; Milne, Thomas A.; Fulga, Tudor A.

doi:10.1101/342485

Cited by 7 publications

(7 citation statements)

References 23 publications

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“…These results are consistent with those reported by Knapp et al (Knapp et al, 2019) showing that tRNAs can generate functional gRNAs that are constitutively and promoter-independently expressed. Knapp et al (2019) designed a tRNA Pro scaffold (Δ, ΔC55A, and ΔC55G) with minimized promoter strength without affecting the processing activity of the tRNA. An interesting addition to our work would be to implement the usage of these tailored tRNAs.…”

Section: B Discussionsupporting

confidence: 93%

“…However, this extra regulation did not lead to efficient control over the activation of the reporter gene. These results are consistent with those reported by Knapp et al (Knapp et al, 2019) showing that tRNAs can generate functional gRNAs that are constitutively and promoter-independently expressed. Knapp et al (2019) designed a tRNA Pro scaffold (Δ, ΔC55A, and ΔC55G) with minimized promoter strength without affecting the processing activity of the tRNA.…”

Section: B Discussionsupporting

confidence: 93%

“…However, most minimal promoters coupled to inducible operators rely on polymerase II (pol-II) transcription (Knapp et al, 2019). As pol-II transcripts are altered with a 5' cap and poly-A tail and exported from the nucleus, the efficiency of a gRNA expressed under pol-II would be highly compromised.…”

Section: Effect Of Grna Regulation On Copper-inducible Dcasev21 Activationmentioning

confidence: 99%

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A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

Garcia-Perez

Diego-Martin

Quijano‐Rubio

et al. 2021

Preprint

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CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

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Section: B Discussionsupporting

confidence: 93%

Section: B Discussionsupporting

confidence: 93%

Section: Effect Of Grna Regulation On Copper-inducible Dcasev21 Activationmentioning

confidence: 99%

See 1 more Smart Citation

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

Garcia-Perez

Diego-Martin

Quijano‐Rubio

et al. 2021

Preprint

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“…S6), the efficiency of multi-gene editing using the PTG strategy was lower than that achieved with the co-injection strategy, likely due to the requirement for simultaneous processing of 4 sgRNAs from a single template. Studies in plants and flies have reported that insertion of a tRNA between the PolIII promoter and the first sgRNA increased transcription efficiency (31), which was found to be due to the additive effect by the innate tRNA's promoter activity (32). We attempted this approach without success (data not shown), an indication of species-specific differences in short RNA processing.…”

Section: Discussionmentioning

confidence: 99%

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

et al. 2022

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Background & Aims: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or peri-zygotic embryos. These approaches are time consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a Rapid In vivo Multiplexed Editing (RIME), and to provide a proof-of-concept of this system.Approach & Results: RIME, a system wherein CRISPR/Cas9 technology, paired with adenoassociated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver.The method is quick, requiring as little as 1 month from conceptualization to knockout (KO), and highly efficient, enabling editing in >95% of target cells. To highlight its utility, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation. Conclusion:RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo..

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“…Pol II promoters naturally transcribe mRNAs while pol III promoters naturally transcribe transfer RNAs and small ribosomal RNAs [168]. Pol II promoters are used for temporal and spatial gene expression while pol III promoters for constitutive gene expression [169]. Types of commonly used pol II promoters include CMV and CAG (which uses CMV as an enhancer) [111] and commonly used pol III promoters include U6, 7SK, and H1 [170].…”

Section: Regulatory Elementsmentioning

confidence: 99%

Mechanisms to investigate the nervous system : genetic engineering with adeno-associated viruses

Torregrosa¹

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Since the discovery of the adeno-associated virus (AAV) in 1965, AAVs have been harnessed as delivery vehicles for targeted transfer of DNA or RNA sequences for gene editing. Depending on the AAV cargo, the gene can systematically manipulate DNA, RNA, or amino acids affecting a cell's natural function adding to our fundamental understanding in systems such as the central and peripheral nervous systems. This research uses AAV delivery for both optogenetics and CRISPR-Cas to 1) engineer an in vitro model to stimulate the sympathetic and parasympathetic neurons connecting to heart muscle cells and 2) validate a technique to quantitatively measure the efficiency and tropism of in vivo central nervous system AAV delivery. This dissertation impacts the field by 1) simplifying and controlling the complex signaling of the ANS-cardiac connection and 2) implementing gene knock out to quantify AAV delivery efficiency. Finally, in the broader impact of my work, I share unorthodox communication experiences during my Ph.D.Optogenetics is a technique to control the cells and tissue regions of the nervous system with light. AAVs can deliver a messenger RNA sequence that codes for a light sensitive ion channel/pump, called an opsin, that allows researchers to control cell depolarization with light stimulation. Optogenetics can control sympathetic and parasympathetic neurons in the autonomic nervous system (ANS) that synapse the heart creating a systematic reaction measured by heartbeat. A simplified ANS-cardiac in vitro model with independent optogenetic control of sympathetic and parasympathetic neurons was engineered to isolate the neuronal contributions to heart muscle cells.An efficient and versatile gene-editing technique exploiting clustered regularly interspaced short palindromic repeats along with a Cas nuclease (CRISPR-Cas) can permanently edit a DNA or RNA sequence. AAVs can deliver CRISPR-Cas sequences targeting a specific gene to knock out resulting in a protein reduction. This protein reduction can be measured and correlated to the AAV delivery efficiency.Not only can this high throughput, quantitative efficiency screen has tissue type resolution but the potential iii for cell-type resolution if the gene codes for a specific cell marker protein. A screen was optimized to measure AAV transduction efficiency in neurons, astrocytes, and oligodendrocytes in the central nervous system. v

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Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

Cited by 7 publications

References 23 publications

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Mechanisms to investigate the nervous system : genetic engineering with adeno-associated viruses

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