Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM.

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

Optimized strategy for in vivo Cas9-activation in Drosophila.

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

CRISPR/Cas9 and Nanotechnology Pertinence in Agricultural Crop Refinement.

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) method is a versatile technique that can be applied in crop refinement. Currently, the main reasons for declining agricultural yield are global warming, low rainfall, biotic and abiotic stresses, in addition to soil fertility issues caused by the use of harmful chemicals as fertilizers/additives. The declining yields can lead to inadequate supply of nutritional food as per global demand. Grains and horticultural crops including fruits, vegetables, and ornamental plants are crucial in sustaining human life. Genomic editing using CRISPR/Cas9 and nanotechnology has numerous advantages in crop development. Improving crop production using transgenic-free CRISPR/Cas9 technology and produced fertilizers, pesticides, and boosters for plants by adopting nanotechnology-based protocols can essentially overcome the universal food scarcity. This review briefly gives an overview on the potential applications of CRISPR/Cas9 and nanotechnology-based methods in developing the cultivation of major agricultural crops. In addition, the limitations and major challenges of genome editing in grains, vegetables, and fruits have been discussed in detail by emphasizing its applications in crop refinement strategy.

Development and Validation of CRISPR Activator Systems for Overexpression of CB1 Receptors in Neurons.

Gene therapy approaches using viral vectors for the overexpression of target genes have been for several years the focus of gene therapy research against neurological disorders. These approaches deliver robust expression of therapeutic genes, but are typically limited to the delivery of single genes and often do not manipulate the expression of the endogenous locus. In the last years, the advent of CRISPR-Cas9 technologies have revolutionized many areas of scientific research by providing novel tools that allow simple and efficient manipulation of endogenous genes. One of the applications of CRISPR-Cas9, termed CRISPRa, based on the use of a nuclease-null Cas9 protein (dCas9) fused to transcriptional activators, enables quick and efficient increase in target endogenous gene expression. CRISPRa approaches are varied, and different alternatives exist with regards to the type of Cas9 protein and transcriptional activator used. Several of these approaches have been successfully used in neurons in vitro and in vivo, but have not been so far extensively applied for the overexpression of genes involved in synaptic transmission. Here we describe the development and application of two different CRISPRa systems, based on single or dual Lentiviral and Adeno-Associated viral vectors and VP64 or VPR transcriptional activators, and demonstrate their efficiency in increasing mRNA and protein expression of the Cnr1 gene, coding for neuronal CB1 receptors. Both approaches were similarly efficient in primary neuronal cultures, and achieved a 2-5-fold increase in Cnr1 expression, but the AAV-based approach was more efficient in vivo. Our dual AAV-based VPR system in particular, based on Staphylococcus aureus dCas9, when injected in the hippocampus, displayed almost complete simultaneous expression of both vectors, high levels of dCas9 expression, and good efficiency in increasing Cnr1 mRNA as measured by in situ hybridization. In addition, we also show significant upregulation of CB1 receptor protein in vivo, which is reflected by an increased ability in reducing neurotransmitter release, as measured by electrophysiology. Our results show that CRISPRa techniques could be successfully used in neurons to target overexpression of genes involved in synaptic transmission, and can potentially represent a next-generation gene therapy approach against neurological disorders.