Abstract

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

Highlights

  • After several decades of development, genome engineering has become a highly developed and indispensable tool for biological study and bioengineering applications [1]

  • The pRC02 plasmid was constructed to test the activity of the RepH protein and rolling circle origin (RCORI) in E. coli

  • We developed a scalable platform for genome editing based on in vivo synthesis of linear single strand DNA (ssDNA) in living cells

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Summary

Introduction

After several decades of development, genome engineering has become a highly developed and indispensable tool for biological study and bioengineering applications [1]. A large toolbox has been developed using different molecular machinery, including recombinases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) systems (CRISPR/Cas system) [2,3,4]. Homologous recombination can introduce precise deletions or insertions into the genome. The donor DNA template, either single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), is a crucial part for homology-directed genome editing [5,6]. Recombineering mediated by linear dsDNA fragments or circular plasmids requires long (1–5 kb) homology arms flanking the insertion sequence [7,8]. Commercial ssDNA oligos can only provide short homology arms (

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