A step by step protocol for genomic knock-in of long sequences using dCas9-SSAP editor

Abstract Gene-editing is a powerful tool for probing the mechanisms of human health and diseases, and holds promise as a durable therapeutic approach. Exemplified by CRISPR-Cas systems, gene-editing tools often cause DNA damage at on- and off-target sites, and thus trigger diverse endogenous DNA repair processes that are error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here, in this protocol, we present a method that couples microbial single-strand annealing proteins (SSAPs) with dCas9-guideRNA complex to stimulate DNA strand exchange for gene-editing. The dCas9-SSAP editor had low editing errors at target loci, minimal detectable off-target effect, and higher accuracy than canonical Cas9 methods. This method was effective for inserting sequences up to kilobase-scale, with up to ~20% knock-in efficiencies and robust performances across donor designs and cell types, including human stem cells.