Information

Date:
5th July 2019
Time:
13:00
Location:
LMS Seminar Room
2nd floor CWB, Faculty of Medicine
Imperial College London
Hammersmith Hospital Campus
Du Cane Road
London
W12 0NN

Ad hoc Seminar – John Hawkins

“High-throughput benchmarking of engineered CRISPR-Cas nucleases”

I will present a new NGS-based platform that measures the cleavage and binding specificity of natural and engineered CRISPR-Cas nucleases. Our new nuclease sequencing pipeline (NucleaSeq) exhaustively measures cleavage kinetics and captures the time-resolved identities of cleaved products for a large library of partially gRNA-matched DNAs. The same DNA library is used to measure the binding specificity of each enzyme via the chip-hybridized association mapping platform (CHAMP). Coupling NucleaSeq and CHAMP, we benchmarked the cleavage and binding specificities of Cas12a and four SpCas9 variants for 105DNAs containing mismatches, insertions, and deletions. Engineered Cas9s dramatically increase cleavage specificity, but provide minimal improvement to overall binding specificity, with Cas9-HF1 performing best. In contrast, Cas12a strongly discriminates along the whole hybridized sequence during binding and cleavage. Surprisingly, both Cas9 and Cas12a produce variable ssDNA overhangs. Initial cleavage position and subsequent end-trimming vary with the nuclease, gRNA sequence, and position and base identity of modified target DNAs. By programming mismatches between gRNA and target DNA, these nucleases can generate incompatible DNA ends without slowing cleavage, ultimately biasing cellular repair outcomes. More broadly, NucleaSeq and CHAMP enable rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural CRISPR-Cas nucleases.

 

John Hawkins

The University of Texas at Austin