Jain Lab
Multiplexing
​Various diseases display similar symptoms, but typically, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics and more traditional forms of diagnostics only test for the presence of a single type of pathogen. Due to the prevalence of HCV in the US, we determined there was a need for a single diagnostic test that could differentiate between HCV subtypes, specifically 1a and 1b. We explored the feasibility of combining multiple Cas (CRISPR-associated protein) orthologs and Cas predecessors based on temperature of trans-cleavage activity, allowing for the detection of up to four targets, without the need for expensive equipment such as a thermocycler or multiple fluorescent channels. To limit cross-over activity and widen the threshold between positive and negative samples, we also incorporated RNA thermometers, such as HSP17, that could block cleavage activity at lower temperatures. With the increasing threat of emergent pathogens, we believe that the multiplexing of Cas orthologs for disease detection can have a broad impact on diagnostics for human health as well as diagnostics of plant and animal pathogens within the agricultural industry.
CODE
Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5’ to 3’ exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.
LEAP
CRISPR-Cas12a enzymes are RNA-guided nucleases widely used for programmable genome editing and diagnostics. Perfect complementarity between guide RNA and target DNA is essential for efficient binding and cleavage of DNA by Cas12a. However, we report that a particular ortholog of Cas12a, AsCas12a, shows an unexpected tolerance to noncomplementary insertions at various positions in its DNA target. AsCas12a remains functional despite DNA bubbles or loops in the crRNA-target DNA duplex, displaying both cis- and transcleavage activities even when the target harbors insertions of lengths 1-20 nt in the crRNAbinding region. This activity is sequence independent and works for ssDNA and is observed on dsDNA in vitro for specific insertion lengths/positions and DNA topologies but is strongly diminished in cells. Among 12 Cas12a orthologs tested, only AsCas12a exhibits this tolerance, making it a unique member of the Cas12a family. Structural analysis suggests a distinctive αhelix in AsCas12a's WED domain is required for this flexibility. Upon deleting this α-helix, AsCas12a loses its ability to tolerate insertions. This discovery can be utilized to detect single nucleotide polymorphisms and enable PAM-flexible DNA cleavage with Cas12a. Our findings expand our fundamental understanding of CRISPR-Cas12a systems. In conclusion, we uncover and characterize a unique property of AsCas12a to tolerate insertions in its target.
Connect with me on LinkedIn!
