Knockin marmoset reporters for non-invasive measuring of genome-editing efficiency

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Project Summary The past decade has seen unprecedented advances in genome editing technologies and the approval of several targeted gene therapies by the Food & Drug Administration. As such, targeted endonuclease-based approaches to gene therapy now hold realistic promise for the widespread treatment and reversal of a large number of human diseases. Although early versions of CRISPR/Cas systems suffered from problems with efficiency and specificity, recent advances achieved through directed-evolution, targeted protein engineering, and the identification of editing enhancers have put widespread use of gene therapy within reach. However, one major hurdle between the basic science and the implementation of safe and effective clinical interventions is a lack of suitable large animal models for pre-clinical testing of new technologies and therapeutic strategies. Here, we propose a strategy to generate two reporter lines in the common marmoset (Callithrix jacchus) that will be used for monitoring of both on- and off-target editing by Cas9 and adenine base editors at single-cell resolution. Large animal reporters must be highly efficient and capable of testing multiple aspects of gene editing if they are to be broadly useful and capable of overcoming the slow sexual maturation and gestational periods of primates. Taking this into consideration, we will utilize well-established fluorescent reporters in conjunction with a newly-developed variant of luciferase called Akaluc that can be used for non-invasive bioluminescent imaging in marmosets with single-cell sensitivity. We will generate an initial set of two marmoset reporters capable of testing knock-in and adenine base editing in a non-invasive manner through on-target editing-mediated activation of Venus-Akaluc expression. Following initial screening for on-target editing via whole-body imaging with an electron-multiplying charge-couple device (EMCCD) camera, tissue can be collected from these reporter animals and used for single-cell analyses of both on- and off-target editing through fluorescence activated cell sorting (FACS) and single-cell sequencing. Our approach establishes a versatile and efficient platform for testing editing technologies in a non-human primate species and can be easily and rapidly adapted to evolving technologies, making it a valuable system for accelerating the development of safe and effective gene therapies.