**2.5. Genome editing**

Genetics relies on the analysis of mutations and the phenotypic variation they cause to correlate precise sequence changes to particular genes of interest. With the help of genetic engineering techniques, desired traits can also be introduced into plants not expressing them naturally. However, the use of genetically modified crops is hindered by health, environmental, and ethical concerns. Genome editing with site-specific nucleases is the most advanced technology for precise and effective genome engineering, which promises to revolutionize applied research for crop improvement [70,71]. It involves the insertion, elimination, or replacement of a fragment of DNA at desired locations in the genome, by using engineered nucleases that create specific double-strand breaks (DSBs) and stimulate cellular DNA repair mechanisms. There are currently four classes of targetable nucleases discovered and bioengineered that are used to create site-specific DSB: zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) RNA-guided nucleases (RGNs), and engineered meganu‐ clease, also known as homing endonucleases [72–75].

tive splice variants; mapping on the reference genome to elaborate the expression level of known, annotated genes; and the differential expression of selected genes between different genotypes. Such studies will become increasingly important for modern breeding programs since especially biotic and abiotic stresses are clearly regulated by different mechanisms other

First experiments were conducted to study the DNA methylation profile on the model plant *Arabidopsis* to study epigenetic changes upon biotic stresses. A whole set of tools were installed and in-house scripts developed to analyze data derived from whole-genome bisulfide (BS) transformation [67]. The BS transformation converts non-methylated cysteine into a uracil and later, after polymerase chain reaction (PCR) amplification, into a thymine, whereas the methylated cysteine remains a cysteine. Since this technique is looking for single-nucleotide events and since the genomic code is "falsified," there is the need for a high-quality reference and specialized mapping strategies and statistics for the methylation calling [68]. The availa‐ bility of a good-quality reference genome sequence of cassava and whole-genome re-sequenc‐ ing of several clones of interest prompted DNA methylation profiling for some relevant cassava varieties. In this pilot study at IITA, currently in progress, the aim is to reveal dynamic methylation events under biotic and abiotic stresses to gain information on possible epigenetic

With the development of NGS noncoding RNA (ncRNA), especially the smaller species became very easy to detect, and many studies demonstrated that these ncRNAs are important players in gene regulation, regulation of DNA and histone methylation, and defense mecha‐ nisms in plants. ncRNA profiles are also important for diagnosing and characterizing virus infections in plants [69]. The virus infection triggers a defense reaction where a cascade of host ncRNA are involved, but also small interfering RNAs (siRNAs) corresponding to the viral genome are found in the plant extract. These endogenous ncRNA and the viral small RNA fragments can easily be detected by NGS. At IITA, we have the expertise and software suite of tools to search and analyze any plant ncRNAs or virus siRNAs. Again biotic and abiotic stresses in plants have a specific profile of expression of different species of ncRNA, and at IITA, we study this phenomenon to create information and tools to improve the breeding

Genetics relies on the analysis of mutations and the phenotypic variation they cause to correlate precise sequence changes to particular genes of interest. With the help of genetic engineering techniques, desired traits can also be introduced into plants not expressing them naturally. However, the use of genetically modified crops is hindered by health, environmental, and ethical concerns. Genome editing with site-specific nucleases is the most advanced technology for precise and effective genome engineering, which promises to revolutionize applied research for crop improvement [70,71]. It involves the insertion, elimination, or replacement of a fragment of DNA at desired locations in the genome, by using engineered nucleases that create specific double-strand breaks (DSBs) and stimulate cellular DNA repair mechanisms. There are currently four classes of targetable nucleases discovered and bioengineered that are

than purely genetic variations.

294 Next Generation Sequencing - Advances, Applications and Challenges

programs.

**2.5. Genome editing**

markers for the next-generation breeding programs.

Over the past few years, all of the above nucleases have been used to create targetspecific mutations in model and crop plants, albeit with some limitations. In all cases, a continuing issue is the delivery of all the reagents efficiently and functionally to the cells or organisms under study. The CRISPR/CRISPR-associated protein 9 (Cas9) tool seems to overcome some of the shortcomings of the other methods [76,77]. Successful examples of targetable nucleases application are reported for *Arabidopsis*, tobacco, rice, maize, soy‐ bean, barley, cabbage, and bunchgrass by using different delivery technologies, including T-DNA plasmid from Agrobacterium, protoplasts and embryonic callus manipulation, and subsequent plant regeneration [70,78–82].

Targetable nucleases are attractive alternative biotechnological tools for trait manipulation and breeding in crop plants. By means of targetable nucleases, mutations can be produced in a very specific manner, and known mutations can be transferred between cultivars or breeding lines without disrupting a favorable genetic background. Although genome editing approaches are relatively new and not yet widely applied, their advantage in terms of safety, robustness, speed, and precision over the classical mutagenesis and breeding is undisputable [75]. Targeted genome editing using artificial nucleases, combined with accurate gene expression analyses, has the potential to accelerate plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner [71] and to restore lost traits through reverse breeding [83]. Although genome editing has not yet been applied to African staple crop species, there is no doubt that this technology will assume a great importance particularly for genetic improvement of asexually propagated crops with limited flowering ability [71].

Furthermore, technologies based on targetable nucleases offer the opportunity to overcome the major concerns of the general public about transgenic crops since the organism with the edited gene do not contain the foreign DNA. In particular, the absence of extra copies of DNAs upon nonhomologous end joining (NHEJ)-mediated gene knockout makes the final plant comparable with those arising from natural mutations. However, the development of dedi‐ cated international legislations is required to effectively promote a wide application of genome editing technologies for crop improvement [70,84]. As knowledge is gained about plant genome organization and gene functions are revealed, the potential of genome editing could be mainstreamed to broaden the genetic base of crops.
