Employing a metagenomic search, we identified a family of miniature CRISPR-Cas type V-L systems capable of RNA-guided dsDNA target cleavage without a tracrRNA. A bacterial depletion screen revealed several active systems which were subsequently shown to have low-level editing in mammalian cells. Among the systems with activity in mammalian cells, we selected a 745aa nuclease effector as a candidate for engineering. An unbiased mutational scanning approach was applied to identify single substitutions that increase indel activity in mammalian cells. Indel-enhancing single substitutions were then screened in combination, with a combinatorially engineered variant demonstrating indel editing activity comparable to SpCas9 in HEK293T cells and exceeding 60% in primary human hepatocytes. Structure determination via cryo-EM revealed the domain organization and nuclease mechanism of the ternary complex. Due to its small size and robust editing levels, this novel miniature CRISPR-Cas type V-L system is an attractive therapeutic candidate for single- or dual-guide excision via all-in-one AAV delivery. 
 
Key Points:
Small size coupled with potent mammalian activity make the combinatorial engineered variant of a miniature type V-L CRISPR-Cas system an attractive candidate for therapeutic genome editing.