Engineering an RNA-based tissue-specific platform for genetic editing through use of a miRNA-enabled Cas12a

Abstract

9The capacity to edit genomes using CRISPR-Cas systems holds immense potential for 10 countless genetic-based diseases. However, one significant impediment preventing broad 11 therapeutic utilization is in vivo delivery. While genetic editing at a single cell level in vitro can be 12 achieved with high efficiency, the capacity to utilize these same biologic tools in a desired tissue 13 in vivo remains challenging. Non-integrating RNA virus-based vectors constitute efficient 14 platforms for transgene expression and surpass several barriers to in vivo delivery. However, 15 the broad tissue tropism of viral vectors raises the concern for off-target effects. Moreover, 16 prolonged expression of the Cas proteins, regardless of delivery method, can accumulate 17 aberrant RNAs leading to unwanted immunological responses. In an effort to circumvent these 18 shortcomings, here we describe a versatile RNA virus-based technology that can achieve cell-19 specific activity and self-inactivation by combining host microRNA (miRNA) biology with the 20 CRISPR-Cas12a RNA-guided nuclease. Exploiting the RNase activity of Cas12a, we generated 21 a vector that self-inactivates upon delivery of Cas12a and an accompanying CRISPR RNA 22 (crRNA). Furthermore, we show that maturation of the crRNA can be made dependent on cell-23 specific miRNAs, which confers cell-specificity. We demonstrate that this genetic editing circuit 24delivers diminished yet sufficient levels of Cas12a to achieve effective genome editing whilst 25 inducing a minimal immunological response. It can also function in a cell-specific manner 26 thereby facilitating in vivo editing and mitigating the risk of unwanted, off-target effects. 27