Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing

Abstract

Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.

TESOGENASE, An Engineered Nuclease Editor for Enhanced Targeted Genome Integration

“…This idea has been verified by multiple studies using different strategies. For instance, physical coupling of dsDNA donor templates together with Cas9 endonuclease by fusing Cas9 together with transcription factors or homologous recombinase such as Rad51, Rad52, POLD3 and the dominant negative form of 53BP1 has been shown to increase dsDNA mediated transgene KI (Gao et al, 2022; Jayavaradhan et al, 2019; Li et al, 2021; Lin-Shiao et al, 2022; Park et al, 2021; Rees et al, 2019; Reint et al, 2021; Shao et al, 2017, 2017; Tran et al, 2019). Other studies engineered the single or double stranded donor DNA with chemical modification such as biotinylation, 5’-triethylene glycol (TEG) modification or covalent conjugation of donor DNA with Cas9 endonuclease (Aird et al, 2018; Carlson-Stevermer et al, 2017; Ghanta et al, 2021; Ma et al, 2017; Savic et al, 2018).…”

Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration

“…This idea has been verified by multiple studies using different strategies. For instance, dsDNA donors have been coupled to Cas9 endonuclease by fusing Cas9 together with DNA binding proteins including transcription factors, recombinase subunits (such as Rad51, Rad52, POLD3) and the dominant negative form of 53BP1 to improve the efficiency of dsDNA knock-in ( Gao et al, 2022 ; Jayavaradhan et al, 2019 ; Li et al, 2021 ; Lin-Shiao et al, 2022 ; Park et al, 2021 ; Rees et al, 2019 ; Reint et al, 2021 ; Shao et al, 2017 , 2017 ; Tran et al, 2019 ). Other studies coupled the single or double stranded donor DNA to Cas9 in vitro using chemical modifications, such as DNA biotinylation for tethering to an avidin-conjugated Cas9 (ref) or 5’-triethylene glycol (TEG) modification or covalent conjugation of donor DNA with Cas9 endonuclease ( Aird et al, 2018 ; Carlson-Stevermer et al, 2017 ; Ghanta et al, 2021 ; Ma et al, 2017 ; Savic et al, 2018 ).…”