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.
This protocol enables rapid, one-step assembly of triple-sgRNA expression cassettes and describes steps for CRISPR knockout sgRNA design, plasmid construction and verification, animal preparation, and neonatal adeno-associated viral vector delivery.
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.
A detailed guide for designing CRISPRi circuits in bacteria, using the CRISPRlator, the first CRISPRi oscillator, as an example that illustrates the design process.
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) a...
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