4. CRISPR/Cas9: a close relationship with TALENs and ZFNs

As previously mentioned, CRISPR/Cas9 system is conformed by a single monomeric protein as well as a complex RNA rearrangement. The Cas9 protein is responsible for carrying out the cleavage process, and a 20-nt fragment corresponding to gRNA is responsible for identifying target sequences [28]. Notwithstanding, the nature of TALENs and ZFNs (zinc finger nucleases) allows them to function as dimers; consequently, protein components are just required to develop the corresponding catalysis processes. A specific domain of the FokI endonuclease performs the cleavage of target sequences. On the other hand, domains corresponding to DNA binding that may be found in different polypeptides are those that confer the sequence specificity [29].

therapeutic applications. On the other hand, since TALENs show repetitive nature, it is usually more complicated to pack and move the molecules into the host cell using viral vectors [31].

In the case of ZFNs, it is important to highlight that in recent years, their use has increased considerably in both industrial and basic research, mainly for the generation of therapeutic adjuvants in both animal and human models. ZFNs comprise arrays of protein fusions with specific binding domains adapted to transcription factors that contain zinc finger complements. In a complementary way, its structure is also conformed by a FokI restriction domain. The zinc finger domains are able to recognize 3–4 bp sequences in the target DNA molecule. Tandem domains (tandem repeats; occurring in the DNA through the repetition of patterns of one or more nucleotides and the position of these is completely adjacent) tend to interlace with nucleotide sequences between 3, 9, and 18 bp length, and this process is repetitive throughout

rate

37.7– 38.5%

12.7– 13.8%

16.4– 19.1%

1.1–5.6% PCR

2.5–2.7% PCR

n.a. Pre-

Detection method

sequencing

sequencing

PCR sequencing

digested PCR

14.5–38% PCR [40]

T7E1 assay

digested PCR

sequencing

n.a. [41]

28.5% PCR + RE [40]

3.2–3.9% PCR + RE [42]

3–8% qPCR and

n.a. Pre-

n.a. [39]

Ref

47

[36]

[36]

[36]

[37, 38]

[14]

[37, 38]

[39]

Species Cas9 codon Trans method Target sequence Promoter Mutation

PEG protoplast RACK1b,

PEG protoplast PDS3, FLS2 CaMV35SPDK,

RACK1c

MPK2, Os02g2382

PEG protoplast PDS3 CaMV35SPDK,

AtU6

AtU6

AtU6

CaMV35S

2xCaMV35S, OsU3

OsU3 or OsU6

PDS, INOX CaMV35S 18–22% PCR

OsU6

TaU6

ZmU3

CaMV35S

PDS CaMv35S,

Co-transfect GFP CaMV35S,

PDS CaMV35S,

CaMV35SPDK, AtU6

Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing

http://dx.doi.org/10.5772/intechopen.81080

Arabidopsis thaliana

Nicotiana benthamiana

N. benthamiana

N. benthamiana

Triticum aestivum

Citrus sinensis Arabidopsis (intron)

Arabidopsis (intron)

Chlamydomonas reinhardtii

Human Leaf

T. aestivum Human Agro-transfect

Leaf

Oryza sativa Rice PEG protoplast PDS, BADH2,

agroinfiltration

agroinfiltration

O. sativa Human PEG protoplast MPK5 CaMV35S,

O. sativa Rice PEG protoplast SWEET14 CaMV35S,

embryo immature

Zea mays Rice PEG protoplast IPK 2xCaMV35S,

Table 1. Examples of plant transient transfection based on CRISPR/Cas9-mediated NHEJ.

cell

infiltration

Human Leaf agro-

Rice PEG protoplast MLO 2xCaMV35S,

A. thaliana Arabidopsis (intron)

Because ZFNs show a necessary interaction with zinc fingers, it is considered that this process is experimentally complicated, and therefore, its application is usually limited within a biotechnological context, especially considering the need for nucleotide sequence specificity. However, TALENs and ZFNs are easy to design, and they are commercially available in most cases at an affordable cost [30]. TALENs can promote homologous recombination at cellular level through repetitive sequences compared to gRNA that is based on a Watson-Crick baseparing principle using target DNA sequences [28, 30]. On the other hand, TALENs and ZFNs are capable of generating DSBs through the restriction of FokI catalytic domains of different overlap sizes that may vary depending on their binding capacity. In comparison, Cas9 has two cleavage domains as previously discussed (i.e., RuvC and HNH). While it is true that ZFNs could target any sequence of interest, this process is subject to the availability of assembly platforms. Today, available molecular libraries can hold up to 100 bp sequences that serve as platforms for functional ZFNs. Thereby, TALEN's targets require thymidine residues to show an efficient functionality, which could limit their application [31].

TALEN protein arrays are exclusive for the group of plant pathogenic bacteria. The repeated sequences comprise 10–20 residue agglomerates that recognize specific DNA molecules. Each repeat has a maximum of 35 amino acids in length, and it is complemented by two adjacent amino acids (RVD, repeat-variable-di-residue) which have the function of conferring specificity to the four nucleotides that structure the DNA strands. In this way, a direct link between repeated sequences and target DNA is observed. Both TALENs and ZFNs are capable of generating DSBs in a specific region of the genome, and as previously discussed, this phenomenon is commonly used to generate gene knock out. RVD codes are used to generate TALEN repeating arrays, and therefore, an affinity of up to 96% can be observed respect to target sequences of interest [31, 32]. It is worth mentioning that TALENs show advantages over ZFNs, for example, TALENs can be extended over any sequence length that is necessary, and for the case of ZFNs, they can only be extended in a range of 9–18 bp, although it is considered that TALENs show less specificity [33].

A disadvantage of TALENs over ZFNs is their considerable size, since the extension of a cDNA sequence encoded by TALENs can be up to 3 kb, while the size per every ZFN is only 1 kb. The considerable size of TALENs could hinder the recombination process at cellular level, and therefore, these arrangements tend to be less attractive for biotechnological processes, mainly in therapeutic applications. On the other hand, since TALENs show repetitive nature, it is usually more complicated to pack and move the molecules into the host cell using viral vectors [31].

4. CRISPR/Cas9: a close relationship with TALENs and ZFNs

46 Transgenic Crops - Emerging Trends and Future Perspectives

an efficient functionality, which could limit their application [31].

that TALENs show less specificity [33].

ficity [29].

As previously mentioned, CRISPR/Cas9 system is conformed by a single monomeric protein as well as a complex RNA rearrangement. The Cas9 protein is responsible for carrying out the cleavage process, and a 20-nt fragment corresponding to gRNA is responsible for identifying target sequences [28]. Notwithstanding, the nature of TALENs and ZFNs (zinc finger nucleases) allows them to function as dimers; consequently, protein components are just required to develop the corresponding catalysis processes. A specific domain of the FokI endonuclease performs the cleavage of target sequences. On the other hand, domains corresponding to DNA binding that may be found in different polypeptides are those that confer the sequence speci-

Because ZFNs show a necessary interaction with zinc fingers, it is considered that this process is experimentally complicated, and therefore, its application is usually limited within a biotechnological context, especially considering the need for nucleotide sequence specificity. However, TALENs and ZFNs are easy to design, and they are commercially available in most cases at an affordable cost [30]. TALENs can promote homologous recombination at cellular level through repetitive sequences compared to gRNA that is based on a Watson-Crick baseparing principle using target DNA sequences [28, 30]. On the other hand, TALENs and ZFNs are capable of generating DSBs through the restriction of FokI catalytic domains of different overlap sizes that may vary depending on their binding capacity. In comparison, Cas9 has two cleavage domains as previously discussed (i.e., RuvC and HNH). While it is true that ZFNs could target any sequence of interest, this process is subject to the availability of assembly platforms. Today, available molecular libraries can hold up to 100 bp sequences that serve as platforms for functional ZFNs. Thereby, TALEN's targets require thymidine residues to show

TALEN protein arrays are exclusive for the group of plant pathogenic bacteria. The repeated sequences comprise 10–20 residue agglomerates that recognize specific DNA molecules. Each repeat has a maximum of 35 amino acids in length, and it is complemented by two adjacent amino acids (RVD, repeat-variable-di-residue) which have the function of conferring specificity to the four nucleotides that structure the DNA strands. In this way, a direct link between repeated sequences and target DNA is observed. Both TALENs and ZFNs are capable of generating DSBs in a specific region of the genome, and as previously discussed, this phenomenon is commonly used to generate gene knock out. RVD codes are used to generate TALEN repeating arrays, and therefore, an affinity of up to 96% can be observed respect to target sequences of interest [31, 32]. It is worth mentioning that TALENs show advantages over ZFNs, for example, TALENs can be extended over any sequence length that is necessary, and for the case of ZFNs, they can only be extended in a range of 9–18 bp, although it is considered

A disadvantage of TALENs over ZFNs is their considerable size, since the extension of a cDNA sequence encoded by TALENs can be up to 3 kb, while the size per every ZFN is only 1 kb. The considerable size of TALENs could hinder the recombination process at cellular level, and therefore, these arrangements tend to be less attractive for biotechnological processes, mainly in In the case of ZFNs, it is important to highlight that in recent years, their use has increased considerably in both industrial and basic research, mainly for the generation of therapeutic adjuvants in both animal and human models. ZFNs comprise arrays of protein fusions with specific binding domains adapted to transcription factors that contain zinc finger complements. In a complementary way, its structure is also conformed by a FokI restriction domain. The zinc finger domains are able to recognize 3–4 bp sequences in the target DNA molecule. Tandem domains (tandem repeats; occurring in the DNA through the repetition of patterns of one or more nucleotides and the position of these is completely adjacent) tend to interlace with nucleotide sequences between 3, 9, and 18 bp length, and this process is repetitive throughout


Table 1. Examples of plant transient transfection based on CRISPR/Cas9-mediated NHEJ.

the entire genome in question. ZFNs act at two sites of the DNA sequence at the cellular level, on the forward and reverse strand, respectively. Since cleavages of specific regions within the genome can be observed, ZFNs are capable to recognize two adjacent sequences, and once the corresponding cleavage occurs, the FokI restriction enzyme domains produce a dimerization prior to the cleavage of the corresponding DNA loci. Thus, DSBs with 5<sup>0</sup> -extensions originate [31, 34, 35]. As listed in Table 1, some examples of experiments related to transient transfection based on CRISPR/Cas9-mediated NHEJ may be cited.

The CRISPR/Cas9 system was also used to investigate the influence of specific genes on the phenotype development in tomato plants [51, 62, 63], as well as to achieve features of agronomic importance, such as delayed ripening of tomato fruit [64] or parthenocarpy [65]. Other members of the Solanaceae family reported to have undergone genetic editing via CRISPR/ Cas9, include tobacco (Nicotiana tabacum) [38, 66], potato (Solanum tuberosum) [67], and petunia

Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing

http://dx.doi.org/10.5772/intechopen.81080

49

The CRISPR/Cas9 technology has also been used to confer molecular immunity against tomato yellow leaf curl virus (TYLCV), using N. benthamiana as host [69], as well as inducing complete resistance to Turnip mosaic virus (TuMV) [70], and improve the stress response in the model

In the case of the emerging oil seed plant, Camelina sativa, the CRISPR/Cas9-targeted genetic edition has improved its fatty acid composition, obtaining a seed oil of superior quality on multiple levels, which besides being healthier, was more stable to oxidation and better suited

In addition to that, this technology has been used to obtain a nontransgenic cucumber strain (Cucumis sativus L.), resistant to cucumber vein yellowing disease, papaya ringspot mosaic virus-W, and zucchini yellow mosaic virus [74], as well as to successfully induce targeted mutagenesis in the Chardonnay grape cultivar that enhanced its endurance to powdery mildew, and to increase the golden delicious apple cultivar resistance to fire blight disease [75].

Besides the aforementioned, other crops in which the CRISPR/Cas9 technology has been optimized include barley (Hordeum vulgare) and Brassica oleracea [76], watermelon (Citrullus lanatus) [77], as well as the nonherbaceous sweet orange (C. sinensis cultivar Valencia) [42] and

Finally, we would like to stress that there is no scientific evidence whatsoever to assume that genetic modifications produced by modern biotechnological tools, such as CRISPR/Cas9, represent a higher health or environmental risk than conventional breeding techniques. However, public distrust caused by genetically modified crops has led to many countries to implement highly strict and costly regulations that make very difficult to successfully commercialize such products. Interestingly, since CRISPR/Cas9 genetic editing does not necessarily implies the incorporation of foreign DNA, according to some interpretations, the existing legislation might not be applicable to this technology. Therefore, the scientifically informed public discus-

In recent years, the progress in the development of new tools for molecular genetic research has been evident since their application by simple, versatile, and efficient experimental techniques. From all the genome edition systems based on the nucleases application, CRISPR/Cas9 is the most friendly and simple method. It is now clear that the utility of this technology for the modification of specific loci is limited only by the interest of the researcher. In coming years,

(Petunia hybrida) [68].

plant A. thaliana [71].

for biofuel production [72, 73].

poplar (Populus tomentosa) [78].

sion of such legal framework is imperative [43, 75, 79].

6. Conclusions and final considerations
