Abstract
The clustered regularly interspersed palindromic repeats (CRISPR) system is a powerful genome-editing tool to modify genomes, virtually in any species. The CRISPR tool has now been utilized in many areas of medical research, including gene therapy. Although several proof-of-concept studies show the feasibility of in vivo gene therapy applications for correcting disease-causing mutations, and new and improved tools are constantly being developed, there are not many choices of suitable reporter models to evaluate genome editor tools and their delivery methods. Here, we developed and validated reporter mouse models containing a single copy of disrupted EGFP (ΔEGFP) via frameshift mutations. We tested several delivery methods for validation of the reporters, and we demonstrated their utility to assess both non-homologous end-joining (NHEJ) and via homology-directed repair (HDR) processes in embryos and in somatic tissues. With the use of the reporters, we also show that hydrodynamic delivery of ribonucleoprotein (RNP) with Streptococcus pyogenes (Sp)Cas9 protein mixed with synthetic guide RNA (gRNA) elicits better genome-editing efficiencies than the plasmid vector-based system in mouse liver. The reporters can also be used for assessing HDR efficiencies of the Acidaminococcus sp. (As)Cas12a nuclease. The results suggest that the ΔEGFP mouse models serve as valuable tools for evaluation of in vivo genome editing.
Highlights
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/clustered regularly interspersed palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9), have emerged as well-established tools for targeted manipulation of the genomic sequence
Microinjection of a solution containing single guide RNA (gRNA) targeting EGFP (Table S1; Figure 1A) and Cas[9] mRNA (SpCas[9], derived from Streptococcus pyogenes) into 108 zygotes derived from the EGFP-pA Tg mouse line (EGFP Tg) produced six live pups after zygote implantation
We confirmed the loss of EGFP fluorescence in several organs (Figures 1D and 1E) and performed fluorescence-activated cell sorting (FACS) analysis of splenic cells to confirm loss of EGFP fluorescence at cellular levels, whereas the fluorescence in the parental line was readily detected (Figure 1F)
Summary
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have emerged as well-established tools for targeted manipulation of the genomic sequence. They are used for insertion and deletion of nucleotides (indels) by non-homologous end joining (NHEJ) and for targeted genome replacement via homol-. Molecular Therapy: Nucleic Acids Vol 24 June 2021 a 2021 The Authors. Our results demonstrate that DEGFP mice serve as valuable tools for in vivo genome editing research
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