TL;DR
This chapter describes how to design spacer sequences and install them into the guide RNA expression plasmid and describes how to mutate the handle of gRNA to achieve tunable knockdown of a target gene.
This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli, and shows how cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression.
Summary CRISPR-Cas9 enables genome editing through the expression of Cas9 and single guide RNAs (sgRNAs). Here, we present a protocol for designing and constructing the vector expressing three sgRNAs targeting a single gene in the mouse brain. We describe steps for CRISPR knockout sgRNA design, plasmid construction and verification, animal preparation, and neonatal adeno-associated viral (AAV) vector delivery. We then detail procedures for brain preparation and immunofluorescence-based validation of gene disruption. This protocol enables rapid, one-step assembly of triple-sgRNA expression cassettes. For complete details on the use and execution of this protocol, please refer to Ogawa et al.1
The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation and makes biomolecular condensation a general principle for enhancing CRISPR gene regulation.
MORTISE is introduced, a compact Cas6f-based RNA system to repress target bacterial genes without relying on host RNA factors, enables multiplex control across bacteria, and supports pathway balancing when genetic knockouts are unsuitable.
Summary Here, we present a CRISPR-Cas9 genome-editing protocol for Saccharomyces cerevisiae that simplifies guide construction and enables rapid guide iteration by replacing restriction/ligation cloning with PCR-based guide installation and seamless Cas9-plasmid recircularization. We describe single-guide RNA (sgRNA) and homology-directed repair (HDR) donor design, guide insertion into a KanMX/G418-selectable Cas9-sgRNA plasmid, plasmid cloning and sequence verification in E. coli, and lithium acetate/polyethylene glycol (LiAc/PEG) co-transformation of yeast with the verified plasmid and HDR donor.
