Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.
Leveraging breadth and depth of the scientific workforce invites creativity, relevance, and differing views that directly tie into innovation and problem solving. The NIH BRAIN Initiative is using a multi-pronged strategy to enhance diversity and inclusion toward promoting the best science.
Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in vivo assays to evaluate and compare the performance of CRISPR knockin reagents for both editing efficiency and precision. We tested combinations of Cas9 and DNA donor template variants and determined that Cas9-CtIP with in situ linearized donors display fold-increases of editing precision in cell lines and in vivo in the mouse brain. Iterating this process, we generated novel compound fusions, including eRad18-Cas9-CtIP that showed further fold-increases in performance. Continued development of precision editing reagents with this platform holds promise for direct in vivo knockin across model organisms and future targeted gene therapies.
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