Abstract
The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA in vivo. In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.
Highlights
Wheat is one of the staple food crops in the world, and currently feeds more than a quarter of the global population (Bushuk, 1997; Gustafson et al, 2009)
This research demonstrates the effectiveness of our simplified protocol for protoplast isolation from wheat leaf tissue, subsequent transformation with CRISPR-Cas9 RNPs and GFP plasmids, and analysis with online deconvolution software
While other protocols are available for protoplast isolation and transformation, there is a need for consensus and clarity in some steps
Summary
Wheat is one of the staple food crops in the world, and currently feeds more than a quarter of the global population (Bushuk, 1997; Gustafson et al, 2009). In response to the dearth of available protoplast isolation and RNP transformation protocols, we have developed a simplified protocol that requires no specialized equipment, results in high viable protoplast yields, and gives comparable editing percentages to other protocols, including those that use plasmids (Figure 1). This protocol was tested on two varieties of hexaploid spring wheat: Bobwhite and Chinese Spring, and three gene targets: GW2-B, PinB-D, and ASN2-A. Each gene target was tested in both varieties a minimum of three times
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