Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma

Abstract

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Low-Temperature Plasma Pretreatment Enhanced Cholesterol Detection in Brain by Desorption Electrospray Ionization-Mass Spectrometry Imaging

“…In neurons, the lipid rafts are also found in synapses where they have a leading role in the pre- and postsynaptic functions . LTP has some characteristic features that can change surface properties to increase membrane permeability, which is supported by a literature report to facilitate the direct insertion of CRISPR/Cas9 into plant cells after LTP treatment . We also observed a distinguished color of the H&E image (Figure a) between LTP-treated and nontreated sections due to eosine absorption efficiency, which might be an effect of LTP treatment.…”

“…47 LTP has some characteristic features that can change surface properties to increase membrane permeability, which is supported by a literature report to facilitate the direct insertion of CRISPR/ Cas9 into plant cells after LTP treatment. 44 We also observed a distinguished color of the H&E image (Figure 2a) between LTP-treated and nontreated sections due to eosine absorption efficiency, which might be an effect of LTP treatment. However, the LTP treatment did not affect the heterogeneity of the tissues because the anatomical regions, including cc and aca are prominent in all sections (Figure 2a and Figure 5a).…”

Mechanisms for introducing 250 kDa fluorescent molecules and Cas9/sgRNA into plant cells by plasma treatment

“…27) They also report the successful introduction of RNP into plant cells for genome editing. 28) However, the introduction mechanism has not been clarified in those reports. In addition, genome editing is only the result of expression analysis by PCR, and there is no fluorescent image of the plants with actual GFP expression, which poses problems in the efficiency of introduction and the success rate of genome editing.…”

Seed storage allergens tackled via next-generation research assistant

“…Modern and innovative approaches of precise genome editing (PGE) methods, well reviewed by Yıldırım et al (2023), enable the modification of specific gene ortions by using catalytically impaired Cas9, allowing the direct writing of new genetic information into a specified DNA site (Andolfo et al, 2016). Recent advancements, including the identification of new Cas variants and the development of new editing strategies, demonstrated the evolving landscape of PGE techniques in plants (Yanagawa et al, 2023;Yıldırım et al, 2023). This would enable us to modify specifically the epitopic sequence instead of silencing the proteins, thus avoiding the risk of pleiotropic effects associated with traditional gene knock-out techniques.…”