New method of targeting gene using CRISPR/cas9 and TALENs
Authors: Gajendra Rathod1, Deepak Pawar2, Rakesh Kumar Prajapat2
1Ph.D scholar, Division of Plant Physiology, IARI, New Delhi-110012
2 Ph.D scholar, NRCPB, IARI, New Delhi-110012

Targeted genome editing is a broadly applicable approach for efficiently modifying essentially any sequence of interest in living cells or organisms. This technology relies on the use of engineered nucleases. Engineered nucleases are artificial proteins composed of customizable sequence-specific DNA- binding domain fused to a nuclease that cleaves DNA in a non-specific manner. These targetable nucleases are

1) Meganucleases derived from microbial mobile genetic elements,

2) Zinc finger (ZF) nucleases based on eukaryotic transcription factors,

3) Transcription activator-like effectors (TALEs) from Xanthomonas bacteria and

4) RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas.

These nucleases induce double strand breaks into specific DNA sites which are then repaired by mechanisms of NHEJ (Non Homologous End Joining) and HDR (Homology Directed Repair). TALENs are fusion proteins derived from the DNA recognition repeats of native or customized TAL effectors fused with the DNA cleavage domains of Fok1, to create site-specific gene modifications in plants and other organisms. Bacteria use CRISPRs (clustered regularly interspaced palindromic repeats) naturally as an anti-viral defense immunity mechanism. In that natural context, CRISPR DNA sequences are transcribed into CRISPR RNAs that in turn can lead to the destruction of very specific viral DNA sequences via the Cas9 nuclease. Nowadays scientist mostly use CRISPR/cas9 and TALENs because of fast and easy nature of this technique but the construction of targeting vectors, targeting efficiency which is dependent on substantial variation in the frequency of homology directed repair system are the constraints of this system, to triumph over this scientist have developed new method which is based upon microhomology mediated end joining (MMEJ). MMEj is a double strand break mechanism that uses microhomologous sequences (5-25bp) for error prone end joining. In the cell cycle MMEJ repair is active during G1/early S, whereas HDR is active during late S/G2.

By using this natural DNA repair mechanism a novel method of MMEJ mediated gene knock in strategy referred to as PITCh (precise integration into targeted chromosome) have been developed which enables efficient targeted integration of large DNA fragments in a wide range of cells and organism even those with low HDR activity. In PITCh method a single pair of TALENs/ (CRISPR/Cas9) and vector containing TALENs/ (CRISPR/Cas9) target site are constructed and cointroduced. For engineering of PITCh vector two TALEN target sites should be added at both ends of the cassette in the PITCh vector, the same TALEN set can be used to target loci on the genome and on the PITCh vector. To generate microhomologous sequences the TALEN/(CRISPR/Cas9) target sites on the PITCh vector should contain a different spacer sequences compared with the original genomic sequences, in which the anterior and posterior half are switched off. The genomic sequence and PITCh vector can be cut by the same TALEN/(CRISPR/Cas9) pair and the linearised PITCh vector contains microhomologous DNA ends corresponding to the genomic cleavage site, After MMEJ dependent integration, the whole vector is precisely incorporated into the genome with two TALEN target sites however these TALEN/(CRISPR/Cas9) target sites are hardly cut by TALEN, because they contained shortened spacer regions which are out of the optimal range for double strand introduction by TALEN/(CRISPR/Cas9).


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