The Kleinstiver lab is focused on developing new and improved gene-editing technologies. During his presentation, Dr. Kleinstiver discussed protein engineering strategies applied by his team to improve on-target activity, targeting range, and specificity/safety ofCRISPR/Cas9 nuclease.
Targeting range is a limitation imposed by protospacer adjacent motif (PAM) sequences on the access of Cas9 nuclease to edit broad genomic sites. Therefore, Dr. Kleinstiver’s team engineered changes in amino acid residues relevant for PAM recognition. In addition, to expedite the screening of Cas9 nuclease libraries, his team developed a high-throughput PAM determination assay for screening (HT-PAMDA) Cas9 PAM specificities. This approach has enabled the Kleinstiver team to identify various forms of Cas9, including SpG (NGN) and later SpRY (NAN), with relaxed PAM requirements.
Cas9 nuclease’s off-target effects limit the therapeutic application of current gene editing tools due to potential unwanted genomic changes. With newly engineered Cas9s having relaxed PAM requirements, a critical question that the Kleinstiver lab had to address was the potential for increased off-target activities. Using a previously developed assay, GUIDE-seq, they confirmed that SpG and SpRY activities targeted an increased number of off-target sites. Introducing high-fidelity changes in SpG and SpRY sequences allowed the reduction of off-target edits, thus improving their safety.
What are some of the benefits of these newly developed Cas9 nucleases? First, Dr. Kleinstiver shared how nucleases with minimal PAM requirement can facilitate base-editing and prime-editing approaches, which require the Cas nuclease to access exact genomic sites. Lastly, he shared how his lab leverages these new enzymes even as molecular tools in vitro, which provide greater flexibility for cloning approaches.
You could find more information about gene & cell engineering here.