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. 2022 Jan 1;12(1):35–47. doi: 10.7150/thno.66466

Figure 4.

Figure 4

circDNA as donors in genome editing. (A) Comparisons of the activity of different DNA donors in HCR using the Traffic Light Reporter Multi-Cas Variant 1 (TLR-MCV1) cassette in human cells. circular ssDNA (cssDNA) and T-lssDNA (lssDNA: linear ssDNA) donor template-mediated HDR efficiency is dose dependent. The graph shows the percentage of GFP-positive cells as a function of increasing cssDNA and T-lssDNA donor DNA in the presence of SpyCas9 and AspCas12a proteins in TLR-MCV1 K562 cells (left) and HEK293T cells (right). Reprinted from 39. (B) RCA-Cas-mediated genome editing in vivo. The mechanism of the production of linear ssDNA. SpCas9-mediated DNA cleavage of the targeted circDNA, in the absence of a protospacer adjacent motif (PAM) sequence. Right image: photographs 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). Reprinted from 40, copyright (2020) The MDPI Publishing Group.