Abstract: Aspects of the disclosure relate to methods and synthetic regulatory systems for more efficient nuclease-mediated homology-directed repair (HDR). In particular, provided herein are methods for more efficient in vivo and in vitro HDR-based gene editing where the methods comprise introducing into a cell a synthetic regulatory system comprising Cas nuclease, guide RNAs (gRNAs) having various lengths and configured to target distinct nucleotide sequences for simultaneous transcriptional repression (or activation) and genome editing via double stranded break and use of a donor nucleic acid molecule as a template for repair.

Abstract: Aspects of the disclosure relate to synthetic regulatory systems composed of a multifunctional clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease and at least two distinct guide RNAs (gRNAs). The synthetic regulatory system modulates cleavage and transcription, including repression and activation, in a mammalian cell such as a human cell.

Abstract: Provided herein, in some embodiments, are modular transcriptional architectures and methods for regulated expression of guide RNAs in cells, such as human cells, which are based on Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems.