Targeted Genome Editing in plants
Authors: Monika Dalal^**, Basavaprabhu L. Patil, Rohini Sreevathsa
National Research Centre on Plant Biotechnology, Pusa, New Delhi-110012
^**Corresponding author e-mail: monika@nrcpb.org

The ability to incorporate useful genes and to modify genes in their chromosomal context has been the aim of biologists and breeders worldwide. This ambition was driven by two factors; elucidation of gene functions and crop improvement. Therefore besides breeding, several techniques for foreign gene transfer in to plant genome were developed. In all these gene transfer techniques, integration of genes occurs at random sites. Moreover there are problems of multicopy insertions, trans-gene silencing etc. which are not desirable. These problems can be eliminated by targeted genome editing. Targeted genome editing refers to precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci.

Partial homology based method for Gene targeting
Gene insertions whether random or targeted, are governed by DNA repair mechanism of the cell. There are two repair mechanisms; homologous recombination (HR) and non-homologous end joining (NHEJ). In homologous recombination, unbroken sister chromatid or homologous chromosome is used as a template to copy the sequence into the break site whereas in NHEJ repairing, the broken ends are joined independent of any template or homology. In case of prokaryotes and lower eukaryotes (such as fungi, yeast), the gene transfer/integration occurs through homologous recombination and hence gene targeting/ editing can be efficiently carried out in these organisms. In plants and animals, NHEJ is most prevalent which means gene insertion is most of the time a random event. This poses problem for gene targeting in higher organisms. The initial efforts for gene targeting through homologous recombination in plants were based on partial homology to the target site (Paszkowski et al 1988) which was successful albeit at very low frequency. The high efficiency of NHEJ makes it difficult to detect gene transfer through homologous recombination which occurs scarcely. Therefore techniques for efficient screening and positive - negative selection were developed to facilitate detection of gene targeting in plants. However this technique is getting superseded by nuclease based targeted gene editing tools.

Nuclease based methods for gene targeting and editing
These techniques utilize nucleases that induce site specific double stranded breaks (DSB). It was found that HR could be activated by induction of DSB in yeast. Therefore to study the effect of DSBs on HR in plant cell, a homing endonuclease (meganuclease) I-SceI of yeast mitochondrial origin was used (Puchta et al 1993). The study showed that I-SceI is able to induce specific DSBs in the substrate plasmids and showed enhanced HR frequency. The use of this nuclease was limited by the prerequisite of having the I-SceI-site which needed the insertion of their recognition sites at low frequency and near target site which is not always feasible in all cases.

Zinc finger nucleases
A major break though came in the form of FokI, a type IIS restriction enzyme, which can be cleaved in to two independent domains with recognition and cleavage activities. It was shown that cleavage specificity can be changed by substituting the recognition domain of the FokI enzyme. The first engineered site specific nuclease was created by linking two different zinc finger proteins to cleavage domain of FokI (Kim et al 1996). The application of zinc finger nucleases (ZFNs) was first shown in Drosophila and later it has been used for various different organisms including plants and animals. The zinc finger employed in ZFNs is known as Cys2His2. A typical zinc finger usually binds 3 bases, therefore to increase the specificity three fingers are used. The FokI domain needs dimerization for its enzymatic activity. Hence two molecules of ZFN bind to their targets DNA sequences and align on opposite strands in reverse orientation. The dimer is aligned in such a way that zinc fingers contact at least 9 residues (three residues/ finger) on opposite strands thereby flanking a 5- 6 bp long sequence within the target sequence. This sequence acts as site for FokI cleavage. The zinc fingers are a large number of structurally diverse proteins including transcription factors, which provide a wide array of sequence specificity available naturally. In addition, new ones can be customized and developed by protein engineering in labs. ZNFs have been successfully used for genome editing in plants such as tobacco (Townsend et al 2009) and Maize (Shukla et al 2009).


TALEN
TALENs consist of customizable transcription activator-like effectors (TALE) DNA binding domains fused with non-specific FokI cleavage domains. TALE proteins have been found in plant pathogenic bacteria especially in Xanthomonas spp. TALEs function as transcription activators and are characterized by a highly conserved and repetitive region of mostly 33 or 34 amino acid segments in the middle of the protein. Repeat monomers differ from each other mainly in amino acid positions 12 and 13 (repeat variable di-residues, RVDs). There is a strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site. By modifying these RVDs, TALEs can be made to bind desired target sequences. TALEN has been successfully demonstrated in crop plants like rice (Li et al 2012) and maize (Liang et al 2014).

CRISPR/Cas system
CRISPR/Cas system originated from bacteria where it provides immunity against foreign nucleic acids from viruses or plasmid DNAs. In this system, non host DNA (20-50bp named as spacer) is integrated in to the host genome between copies of short repeat sequences known as CRISPR (clustered regularly interspaced short palindromic repeat). On subsequent invasion these spaces transcribe in to small non coding RNAs (crRNA) which consists of the sequence transcribed from the non host DNA (protospacer) sequence, and part of the CRISPR repeat. The crRNA hybridizes with the trans activating CRISPR RNA (tracrRNA) . These crRNA/ tracrRNA complex with the Cas (CRISPR associated) nuclease which degrades the non host DNA or RNA in a sequence specific manner. The type II CRISPR system from S. pyogenes that has been adapted for targeted genome editing consists of three components; crRNA, tracrRNA and Cas9. In the improved technique, the crRNA and tracrRNA are fused into a single guide RNA (synthetic guide RNA, sgRNA). The sgRNA together with Cas9 induce DSBs in target provided they are immediately 5' to protospacer adjacent motifs (PAM sequence, 5'-NGG). In plant system, this technique has been demonstrated for Arabidopsis, tobacco, rice, sorghum, wheat and maize (Jiang et al 2013, Shan et al 2013, Liang et al 2014).

The potential of the nuclease based technique is evident from the rapid improvements in the technique that are being made to enhance its efficiency, performance, and reduce the cost of targeted gene editing. These nuclease based techniques are widely being used in the field of agriculture, pharmaceuticals, creation of transgenic organisms and gene therapies etc.

Suggested reading:
1. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41(20): e188.

2. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 93 :1156-60.

3. Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol 30, 390-392.

4. Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7: 4021-4026.

5. Puchta H, Dujon B, Hohn B (1993) Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res 21: 5034-5040.

6. Shan Q, Wang Y, Li J, Zhang Y, Chen K, et al. 2013. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31:686-88.

7. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, et al. (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437-41.

8. Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, et al. (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459:442-45.

9. Liang Z, Zhang K, Chen K, Gao C (2014) Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System. J Genet Genom 41: 63 e68.

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