A modular system for murine CRISPR genome and epigenome editing

Project Summary RNA-guided DNA sequence recognition by CRISPR/Cas9 has been exploited to develop an array of genome and epigenome editors, including base editors, primer editors, CRISPR-activators (CRISPR-a) and CRISPR-interference (CRISPR-i). These editors consist of either nuclease dead (dCas9) or DNA-nickase (nCas9) variants of Cas9 fused at N and C termini to “accessory protein” which contain the editor-specific activity. The enzymatic activities of these accessory proteins are being evolved very rapidly to increase the accuracy, efficiency, and types of editing possible. Mice harboring these editors as transgenes are invaluable research tools. However, generating mice containing transgenes that express the latest suite of Cas9 editors is challenging, given their rapid rate of change and ever-increasing number. These editors can be delivered using adeno-associated virus (AAV) vectors, but due to packaging limits of AAV most require a dual AAV strategy involving intein-mediated protein splicing, which hampers editing efficiency and homogeneity. Here we propose to generate mouse lines that express the core dCas9 or nCas9 RNA-guided DNA sequence recognition platforms, functionalized at N- and C-termini with protein ligation tags. The accessory proteins and guide RNAs will be introduced using single AAV vectors. This combination will achieve higher level and more homogeneous editing than possible with AAV-only strategies, while retaining flexibility and the ability to exploit the latest advances in editor enzyme engineering. In Aim 1, we will create Cre-triggered mouse/AAV systems for CRISPR-i/a. We will create mice with Cre-activated expression of dCas9 with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered N- or C-terminal epigenetic editors to implement CRISPR-i/a. In Aim 2, we will create Cre-triggered mouse/AAV systems for base and prime editing. We will create mice with Cre-activated expression of nCas9T (nicks the target strand) with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered N- or C-terminal base editors. We will further create mice with Cre- activated expression of nCas9NT (nicks the non-target strand) with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered reverse transcriptase. These mouse/AAV systems will be invaluable reagents to probe disease biology, perform proof-of-concept therapeutic gene editing, and enable efficient in vivo CRISPR screens using genome or epigenome editing. This proposal is responsive to PAR-21-167. Development of Animal Models and Related Biological Materials for Research.