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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

Toolgen

ToolGen

The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar  and apple cultivar such as  fruit crop plants for efficient targeted mutagenesis. We targeted , a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and , and  in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins.

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.

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

Tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib are effective against lung adenocarcinomas harboring epidermal growth factor receptor (EGFR) mutations. However, cancer cells can develop resistance to these agents with prolonged exposure; in over 50% of cases, this is attributable to the EGFR T790M mutation. Moreover, additional resistance mutations can arise with the use of new drugs. Cancer cell lines with specific mutations can enable the study of resistance mechanisms. In this study, we introduced the EGFR T790M mutation into the PC9 human lung cancer cell line-which has a deletion in exon 19 of the EGFR gene-by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9-mediated genome editing. EGFR pyrosequencing and peptide nucleic acid clamping revealed that PC9 cells with EGFR T790M generated by CRISPR/Cas 9 had a higher T790M mutation rate than those with the same mutation generated by long-term exposure to gefitinib (PC9-G); moreover, resistance to gefitinib in these clones was higher than that in PC9-G cells. The clones were also highly sensitive to the 3rd-generation EGFR TKI AZD9291, which is cytotoxic to lung cancer cells with EGFR T790M. The CRISPR/Cas9 programmable nuclease system can be used to generate various cancer cell lines with specific mutations that can facilitate studies on resistance mechanisms and drug efficacy.

Generation of lung cancer cell lines harboring EGFR T790M mutation by CRISPR/Cas9-mediated genome editing.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

Min-hee Jung

Toolgen

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution