CRISPR’ing Wheat with a Gene Gun

Biolistic transfection, also known as particle bombardment, is exactly what it sounds like: microparticles coated with nucleic acids are shot into cells with devices called “gene guns.” The conception of biolistic transfection is credited to Dr. John Sanford, who showed that RNA and DNA shot into onion tissue was transiently expressed and that many onion cells survived the process (provided they were not pelleted by too many microparticles).¹

Why use gene guns? The key advantage of biolistic transfection is that nucleic acid uptake is practically guaranteed for all kinds of cell types and tissues. Unlike chemical transfection, which relies on mediating biochemical interactions with the cell membrane for promoting nucleic acid uptake, biolistic transfection is a physical method capable of penetrating cellular barriers directly. Interestingly, researchers have discovered that using TransIT®-2020 (a chemical transfection reagent) in conjunction with biolistic transfection can increase transfection efficiency in plant cells.²

Note: it remains untested whether TransIT® reagents other than TransIT®-2020, e.g. TransIT-X2®, can also enhance biolistic transfection efficiency.

Below, we SNiP a study that optimized biolistic transfection of Cas9 ribonucleoprotein complexes (RNPs) in Triticum aestivum, i.e. common wheat, which is an important food crop worldwide. Poddar et al. show incubating wheat embryos at the elevated temperature of 30°C for 16 hours post-bombardment improved editing efficiency. They also found that Cas9 was still present in plant cells weeks after bombardment, which highlights the importance of screening for edited cells much later than is commonly reported to obtain a higher number of edited cells.

Depending on the sgRNA used, Poddar et al. found 10-40% of the embryos had the knockout mutation. Because the embryos are totipotent, they could be grown to full plants. To show the utility of the method, they demonstrate that they were able to engineer wheat plants that were protected from a major fungal pathogen of wheat.

Citation

Title: Impact of temperature and time on DNA-free Cas9-ribonucleoprotein mediated gene editing in wheat protoplasts and immature embryos
Authors: Snigdha Poddar, Jaclyn Tanaka et al.
Journal: bioRxiv, 06 April 2022.
DOI: 10.1101/2022.04.05.487229
Product Usage: Immature wheat embryos were bombarded with gold particles that were coated with TransIT®-2020 Transfection Reagent in complex with Cas9 RNPs. Briefly, a 40 µl mixture containing 12.8 µg Cas9 and 6.4 µg sgRNA, was mixed with 20 µl of 0.6-micron gold particles (10 µg/µl) and 1 µl of TransIT®-2020. After incubating on ice for 20 minutes, the particles were pelleted and resuspended in 20 µl of water. The particles were air-dried onto macrocarrier discs and subsequently used for particle bombardment.

Visit the Citations Database to read more primary literature featuring CRISPR-mediated genome editing.