Gene editing with a multi-effector CRISPR Type I system
Use of Cascade holds promise for enhanced binding and reduced off target effects.
Over the past several years, CRISPR technology has proven to be a simple yet powerful tool for editing genomes. Advancements are enabling easy modification of DNA sequences and gene function. Potential applications include correcting genetic defects, treating and preventing the spread of diseases, and improving crops.
Six main CRISPR system types (Types I to VI) and at least 32 distinct subtypes have been identified to date. Each use short CRISPR-derived RNAs (crRNAs) to target invading nucleic acid and many of these nucleic acid targeting systems rely on sophisticated multi-subunit complexes. For example, the CRISPR-associated complex for antiviral defense (Cascade) is a Type I-E system composed of 11 protein subunits and a crRNA complex that relies on complementary pairing between the crRNA-guide and a target nucleic acid sequence, which occurs over 32 nucleotides. Type II systems, however, rely on a single protein (Cas9) and a 20-nucleotide sequence in recognizing invading DNA. Due to its relative simplicity, the Cas9 system has been used for commercial and research purposes in genetic engineering. However, off-target nuclease activity remains an issue and may limit the use of these tools for certain applications.
Image: Cascade (image credit: Boghog)
Differing from the Cas9 approach, the Type I systems rely on a greater number of nucleotides for target DNA recognition and advantageously employ a locking mechanism during target binding. These qualities show promise towards a gene modification device with enhanced specificity in target recognition compared to Cas9 systems. Still, complexity of the Type I CRISPR complex, the multiple reading frames and the delivery of these systems are hurdles to the use of Type I CRISPR complexes as a viable genome editing technology.
Putting the above complexity to practical work, Dr. Blake Wiedenheft and colleagues at Montana State University have developed constructs and methods that permit assembly of a Cascade complex with one or more effector molecules. These Type I CRISPR complexes include multiple copies of some components (such as Cas7 (6 copies) and Cse2 (2 copies) in Cascade with multiple effector molecules. By providing such “multivalent” effector molecules, the complexes and methods can increase efficiency of gene modulation (such as modifying gene expression or base editing) or increase signal strength in the case of reporters or tags.
- Appropriate effector molecules include transcriptional activators and repressors, reporters, and base editors
- Stronger binding
- Less risk of off target effects
- PCT filed (PCT/US2019/059098)