Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

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.

Circular Single-Stranded DNA: Discovery, Biological Effects, and Applications

“…The RNA-guided Cas9 nucleases are from the prokaryotic CRISPR-Cas genome editing system and are used to edit targeted genomes in eukaryotic cells . The donor DNA template is a vital part of homology-directed genome editing, including dsDNA and ssDNA . Recently, the use of long ssDNA templates as donors for homology-directed repair (HDR) has been demonstrated to exhibit lower cytotoxicity and higher efficiency compared with dsDNAs. , Unlike those studies that used linear ssDNA, Iyer et al first evaluated the use of CssDNA derived from phagemids as DNA donors for HDR-mediated targeted integration of DNA in mammalian cells.…”

“…Overall, the CssDNA HDR templates exhibit superior performance compared to other forms of DNA donor templates due to their enhanced HDR efficiency, high specificity, large length capacity, and low cytotoxicity. Surprisingly, Hao et al used CssDNAs derived from RCR plasmid intermediate to perform genome editing in Escherichia coli. This innovative system offers a novel method for dynamic genome editing by linearizing CssDNAs to increase the concentration of linear DNA donors.…”

Single-stranded circular DNA theranostics

“…These data demonstrate that circDNA donors provide an efficient, cost-effective method to achieve knock-ins via CRISPR-Cas gene editing in mammalian cell lines. In another example, Qi et al utilized RCR- and Cas9- mediated in vivo ssDNA synthesis (Figure 4 B) 40 . A single-gene RCR from Gram-negative bacteria was engineered to produce single-stranded circDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli .…”

“…Passage 1, the first round of culture for introducing the allele substitution (11 bp substitution in the lacZ gene); Passage 10, the tenth round of culture for introducing the allele substitution (11bp substitution in the LacZ gene). Reprinted from 40 , copyright (2020) The MDPI Publishing Group.…”