"Opportunities multiply as they are seized."                                                    
- Sun Tzu

Forging Tools

Science is increasingly constrained by the limitations of available technologies.  By developing improved tools and methods, we can catalyze the research of entire fields. The most useful tools expand the scope of existing or emerging technologies, enabling our accumulated expertise to address previously intractable problems.

Our tool development projects focus on novel methods of editing and regulating genomes.

Many of our projects involve the RNA-guided Cas9 nuclease, which can be directed to site-specifically bind and cut almost any sequence by simply expressing a matching RNA.  This is supremely useful in nearly all organisms and essential for facile genome engineering in multicellular eukaryotes.  Except for cases in which our larger goal requires it, we no longer work on direct Cas9 improvements given the intense interest from many other technology development labs.  However, it is so useful that many of our other projects apply it for a variety of purposes - including building safeguards for gene drives capable of spreading genome alterations made with Cas9 through populations.

A major area of focus involves ways of multiplexing CRISPR in an evolutionarily stable manner.  This is challenging because both strings of guide RNAs and natural CRISPR arrays contain highly repetitive sequences that are prone to internal recombination and rearrangement.  We are exploring ways of diversifying these sequences to enable highly multiplex CRISPR.

Though now a touch dated, our Review in Molecular Systems Biology provides a comprehensive background of currently available tools and technologies for genome-scale engineering, while our Perspective in Nature Methods discusses the utility of Cas9 specifically.



Figure 1.  The RNA-guided Cas9 nuclease from S. pyogenes will cut any target DNA sequence, called a "protospacer", that has a "protospacer-adjacent motif" (PAM) matching "NGG", but only when it is guided by an appropriate "guide RNA" (sgRNA) containing a spacer that matches the protospacer sequence.  Cas9 cuts both strands between the third and fourth bases using separate nuclease domains.  Inactivating one of these domains renders it a nickase.  Inactivating both makes it an RNA-guided DNA-binding protein, which can be used to localize other biomolecules to any targeted protospacer.  Other Cas9 proteins from different bacteria recognize different PAMs but are similarly targetable.  Many of these are orthogonal to one another, allowing them to be targeted independently.