Methods for Engineering a Light-Switchable CRISPR-Cas13 System

Abstract

CRISPR-Cas systems have emerged as a highly promising biotechnological tool in recent years. The Cas13 subfamily of CRISPR/Cas proteins act differently than the usual CRISPR-Cas9 system by cutting RNA, rather than DNA. This offers a promising approach to regulate transcript abundance without permanent genetic modification, with applications in both basic science and therapeutics. Here, in collaboration with the Myhrvold laboratory, we propose and test two methods for engineering a light-switchable Cas13 to control RNA cutting precisely over time and in specific cells in a tissue. The first of these methods is the addition of a stem loop to the Cas13 gRNA that can bind the light-activated PAL protein in light conditions, potentially blocking Cas13-gRNA binding. We discovered that modifications to the gRNA can be made without blocking Cas13 function on its own, however the addition of the PAL protein is not capable of conferring light-switchable properties onto Cas13 using the current system design and with PAL binding protein expressed at the tested levels. The second of these methods is the insertion of a light-switchable LOV2 domain at different sites in the cas13 sequence, following recent successes in engineering light-switchable nanobodies and monobodies, which can be followed by a high-throughput screening to test for light-switchable properties. These results indicate that insertion of the LOV2 domain throughout the CAS13 gene can be performed using transposition followed by a procedure known as Golden Gate Cloning. However, modifications to the procedure must be made so that successful insertions can be isolated and tested for light-switchable properties. These tests serve as early evidence that engineering a light-switchable CRISPR-Cas13 is possible, laying the groundwork for a procedure that will allow for tightly controlled degradation of any user-defined RNA in space and time.