In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

By Eunji Kim, T. Koo, S. W. Park, Daesik Kim, Kyoungmi Kim, Hee-Yeon Cho, D. Song, K. J. Lee, M. Jung, Seokjoong Kim and 3 more. 2017

681 citations recorded by the source corpus.

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration. The amount of genetic material that can be packaged in AAV vectors used for genome editing is limited. Here the authors show that the smallest known Cas9 orthologue, cjCas9, can be packaged in a single AAV vector along with sgRNA and a marker gene, and demonstrate efficient gene editing in mice.

Subfields: Molecular Biology

Citations in the corpus

• Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, neutral): “Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Tables 8, 9, 11, 12, 15, and 16.”
• The next generation of CRISPR–Cas technologies and applications (2019, neutral): “) and Campylobacter jejuni Cas9 (ReF.(31)).”
• Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, neutral): “Viral Delivery of dCasRx to a Neuronal Model of Frontotemporal Dementia The Cas13d family averages 930 amino acids in length, in contrast to Cas9 ( 1,100 aa to 1,400 aa depending on subtype, with compact outliers such as CjCas9 or SaCas9), Cas13a (1,250 aa), Cas13b (1,150 aa), and Cas13c (1,120 aa) ”