Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants

Abstract

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

“…CRISPR technologies have advanced rapidly in recent years and hold promise for altering gene sequence and expression in many systems including plants ( Baltes and Voytas, 2015;Luo et al, 2016). Although producing stable germ-line whole-plant mutants has proven challenging in some species, including Arabidopsis (Fauser et al, 2014;Feng et al, 2014), we were able to produce stable mutant lines with high efficiency.…”

The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology

“…In order to achieve CRISPR-mediated HR the DNA template is normally introduced via the biolistic method to increase its copy number in the host (Baysal et al, 2016). As for maize, but involving a higher number of studies, protoplast transient assay is becoming an efficient tool for testing CRISPR-target before starting the transformation of embryos or scutellum derived calli by Agrobacterium or particle bombardment (Gao et al, 2013; Jiang et al, 2013; Xie and Yang, 2013; Zhou et al, 2014; Lowder et al, 2015; Li et al, 2016; Luo et al, 2016; Wang et al, 2016). Regeneration of rice protoplasts is still very challenging, but important optimization efforts may render it feasible in the near future.…”

Making complex things simpler: modern tools to edit the plant genome

“…Фактически, наличие последовательности PAM -это единственное ограничение при выборе сайта для редактирования геномной ДНК с помощью техно логии CRISPR/Cas. Анализ секвенированных геномов растений показал наличие хотя бы одного уникального сайта для редактирования в кодирующих областях не менее чем 90 % генов (Luo et al, 2016), за исключением кукурузы, в которой только 30 % генов содержат такой сайт (Xie et al, 2014).…”

“…К недостаткам использования кон ститутивных промоторов относятся высокая вероятность получения химерных растений, содержащих различные варианты модификации целевой последовательности в геноме, а также низкая эффективность передачи модифи кации в последующее поколение при семенном размно жении . Кроме того, с использованием конститутивного промотора для гена Cas9 обычно не удавалось добиться высокой эффективности редактиро вания при трансформации растений методом погружения соцветий, что, по всей вероятности, объясняется малой активностью этих промоторов в зародышевых тканях (Luo et al, 2016). Эта проблема была решена применением тка неспецифичных промоторов, обеспечивающих высокий уровень экспрессии Cas9 в зародышевых клетках (рис.…”