A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas Ribonucleoprotein Complexes
A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas Ribonucleoprotein Complexes
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DOI：
10.3389/fpls.2020.00769
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发表时间：
2020-06-10
影响因子：
5.6
通讯作者：
Zemetra, Robert S.
中科院分区：
文献类型：
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作者：
Brandt, Kali M.;Gunn, Hilary;Zemetra, Robert S.
基因工程方法CRISPR被认为是一种高效、廉价、易于使用和有针对性的基因改造技术，被广泛认为有潜力解决现在和未来农业面临的许多问题。然而，与所有新技术一样，它也并非没有挑战。预测和检测最困难的挑战之一是由于特定位置的染色质状态而无法访问的基因目标。目前在设计 sgRNA 靶标的过程中无法预测这一点，在花费时间和资源进行完全转化之前检测到这一问题的唯一方法是测试 sgRNA 的体内切割能力。在小麦中，这可以通过原生质体分离和 Cas9 核糖核蛋白复合物的 PEG 转化来实现。因此，我们开发了一种简化的方案来测试小麦中 sgRNA 靶标的可及性。第一步包括在酶溶液中消化小麦叶组织，然后使用过滤器和蔗糖梯度分离可行的原生质体。然后使用 Cas9 核糖核蛋白复合物通过 PEG 介导的转化来转化原生质体。从 CRISPR-Cas 编辑的原生质体中分离 DNA，并进行 PCR 来扩增基因目标区域。然后使用 PCR 产物通过 Sanger 测序评估所选 sgRNA 的编辑效率。这种使用 Cas9 核糖核蛋白复合物分离和转化小麦原生质体细胞的简化方案可快速、轻松地测试体内 sgRNA 可及性，从而简化了 CRISPR 转化项目。
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 sgRNAin 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 accessibilityin vivo.