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. 2020 Feb 18;9(2):467.
doi: 10.3390/cells9020467.

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

Min Hao  1   2   3 , Zhaoguan Wang  1   2   3 , Hongyan Qiao  1   2   3 , Peng Yin  1   2   3 , Jianjun Qiao  1   2   3 , Hao Qi  1   2   3
Affiliations

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

Min Hao et al. Cells. .

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.

Keywords: PAM-independent; SpyCas9; guide RNA; rolling circle origin; rolling circle replication; single-strand DNA.

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Conflict of interest statement

H.Q. is the inventor of one patent application for the biochemical method described in this article. The initial filings were assigned Chinese patent application 201911382846.5. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1
The mechanism for rolling circle origin (RCORI)-mediated DNA synthesis in Escherichia coli. (A) Schematic of circular single strand DNA (ssDNA) production in vivo. The RCR initiator protein RepH, which acts as a dimer, binds to the RCORI through a sequence-specific interaction. This is followed by nicking of the RCORI by initiator protein (RepH), unwinding of the DNA by helicase, and binding of the single-strand DNA-binding protein (SSB) to the displaced leading strand. Once the replication fork reaches the termination site, the free monomer of RepH cleaves the displaced ssDNA. A series of regulation and cleavage events follow, resulting in the release of a circular ssDNA (displaced leading strand). Solid lines represent double-stranded DNA and dashed lines represent single-stranded DNA. (B) Construction of the small plasmid. RCORI105, the RCR origin, sequence consist of 105 bp from pC194; RCORI36, the 36 bp RCR termination sequence from pC194; AmpR, ampicillin resistance cassette; ColEI, high-copy-number ColE1 origin of replication; RCORI75, the newly formed RCORI after circular ssDNA release. (C) Generation of the small plasmid pRC03. pRC01 is a control group which not including RCORI36 to verify small plasmid construction. The arrows show the band of the small plasmid. ScaI was applied to verify plasmid length. Lane M, DNA Marker. (D) Validation of the small plasmid after recovery. -, without ScaI; +, with ScaI. The full sequence of the small plasmid is shown in Table A2.
Figure 2
Figure 2
Design of genetic parts for circular ssDNA production in vivo (A) Schematic for the conversion of the customized gene to ssDNA. The main RCR sequence contains the repH gene, the desired ssDNA sequence and the RCORI gene. Solid lines represent double-stranded DNA and dashed lines represent single-stranded DNA. (B) The structure of pRC14 and pRC17 used to produce circular ssDNA in vivo. The only difference between these two plasmids is the RCR terminator, which is RCORI36 in pRC14 and RCORI65 in pRC17. The arrows represent the designed amplification primers of the circular ssDNA. (C) The terminator strengths of RCORI36 and RCORI65. The circular ssDNA yield was measured by comparing the abundance of the corresponding PCR products. CLS, cells lysed using a lytic buffer; Kit, plasmids extracted using a commercial miniprep kit; the arrow shows the PCR product (1978 bp) of the circular ssDNA. (D) Different combinations of the RCR system. Lane M, DNA Marker (E) The impact of different combinations on the circular ssDNA yield and the effect of RCORI65 as an RCR origin. (a) The expected PCR product for testing the necessary components to produce ssDNA is 1978 bp. (b) The expected PCR product for testing RCORI65 as an RCR origin is 778 bp. The plasmid pRC17 is a control containing all of the RCR components.
Figure 3
Figure 3
Verification and separation of circular ssDNA. (A) The mechanism of the probe binding with the target sequence. Probe 1 is complementary to the circular ssDNA and the control group; probe 2 is complementary to the plasmid. (B) Fluorescence images of the probe binding with DNA from the cell lysate. Lane C, probe 1 + control; Lane P1, probe 1 + cell lysate; Lane P2, probe 2 + cell lysate; Lane M, DNA Marker (C) Schematic of the ssDNA recovery using Magrose Strep-Tactin beads. (D) Comparison of the circular ssDNA recovery yield from cell lysate. Data are shown for the production of circular ssDNA under culture conditions for plasmid extraction. The arrow indicates the band of the circular ssDNA. (E) PCR analysis of the recovered circular ssDNA. The arrow shows the PCR products (1978 bp) of the circular ssDNA.
Figure 4
Figure 4
RC-Cas-mediated genome editing in vivo. (A) The mechanism of the production of linear ssDNA. SpCas9-mediated DNA cleavage of the targeted circular ssDNA, in the absence of a protospacer adjacent motif (PAM) sequence. (B) Verification of the allelic replacement efficiency. There was no significant difference (p = 0.23) in the replacement efficiency between donor 1 and 2 (around 12%) (C) The plasmids with different components used for gene editing. pRC22 includes the RCR system, gRNA and donor DNA; pRC20 includes the RCR system and donor DNA; pRC11 only includes the donor DNA (D) Photograph of the plates showing the effect of linear ssDNA synthesis for gene editing using pRC22. 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). (E) The efficiency of substituting 11 bp in the LacZ gene using the linear ssDNA donor (F) Verification of the insertion efficiency of 1011 bp. (a) The mechanism of the 1011 bp insertion (b) Sanger sequencing of the 1011 bp insertion.
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