Guide RNA Design For Cas-CLOVER

Welcome to our comprehensive tutorial on designing and constructing Cas-CLOVER guides. Cas-CLOVER is a versatile tool used in genome engineering, and this tutorial will walk you through the process of designing guide RNA pairs and constructing them for use with Cas-CLOVER. Whether you're new to genome engineering or looking to refine your skills, this tutorial will provide you with the knowledge and tools needed to effectively utilize Cas-CLOVER in your research.

Part 1: Designing Guide RNA Pairs

1. Understanding the Basics: Learn about the fundamentals of Cas-CLOVER and its applications in genome engineering. Familiarize yourself with the components required for designing guide RNA pairs.

2. Utilizing Design Tools: Explore open-source gRNA design tools such as CRISPOR and CHOPCHOP. Understand how these tools facilitate the design of guide RNA pairs for Cas-CLOVER.

3. Setting Parameters: Learn about essential parameters for designing guide RNA pairs, including GC content, efficiency, and location. Understand how to filter potential gRNAs based on these parameters to ensure optimal performance.

4. Manually Designing sgRNA Pairs: Discover the process of manually designing sgRNA pairs for Cas-CLOVER using molecular cloning software. Learn how to identify target sequences and annotate guide RNA pairs effectively.

Part 2: Constructing Cas-CLOVER Guides

1. Synthetic Guide Synthesis: Explore options for ordering synthetic guides from providers like Synthego. Understand the process of converting DNA target sequences to RNA and assembling guides for use with Cas-CLOVER.

2. Plasmid-Based sgRNA Expression: Learn about plasmid-based sgRNA expression and the process of ordering oligos for guide sequences. Understand the assembly of guide RNA pairs via Golden Gate Assembly (GGA) and cloning into destination plasmids.

3. Golden Gate Assembly (GGA): Explore the components of the GGA destination plasmid and understand its role in guide RNA assembly. Learn the step-by-step process of Golden Gate Assembly for inserting guide RNA pairs into the base vector.

4. Cloning the Guide Cassette: Understand the final steps of cloning the entire cassette via HiFi cloning into the desired plasmid. Learn about variations in assembly techniques depending on destination plasmids.