BioProject

Programmable gene integration (PGI) technologies are an emerging modality withessential for exciting applications toin both basic research and translational development. More...

Programmable gene integration (PGI) technologies are an emerging modality withessential for exciting applications toin both basic research and translational development. Programmable addition via site-specific targeting elements (PASTE) is a PGI method enabling precise and efficient programmable integration of large DNA sequences into the genome. PASTE offers improved editing efficiency, purity and reprogrammability compared to previous methods for long insertions into the mammalian genome. By combining the specificity and cargo size capabilities of the site specific integrases with the programmability of prime editingCRISPR-based approaches, PASTE can precisely insert cargoes as large as at least 36 kb with high efficiency. Here, we outline the best practices for design, optimization, execution and analysis of PASTE experiments and recommendations at each step. We outline an example PASTE experiment for integration of EGFP at the human NOLC1 and ACTB genomic locilocus as well as the materials needed to read out and analyze the experiment by next generation sequencing (NGS) orand droplet digital PCR (ddPCR). We have included guidelines for designing and optimizing a custom PASTE experiment for integration of desired payloads at alternativeany genomic loci, as well as guidelines for guide optimization, in-frame protein tagging, and multiplexed insertions. To facilitate setup of experiments, we provide sequences, plasmids, and guidance for the delivery of PASTE components via viral vectors or in vitro transcribed RNA. Most experiments in this protocol can be performed in as little as 2 weeks, allowing for precise and versatile programmable gene insertion. Less...