Essay Example on Comparing Zinc Finger Nucleases and Transcription Activator








Summary Comparing Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases for Gene Targeting in Drosophila Beumer et al 2013 G3 Genes Genomes Genetics 3 1717 25 The aim of the study was to investigate the use of Transcription activator like effector nucleases TALENs in genome editing by comparing their effectiveness as compared to the widely used Zinc finger nucleases ZFNs Both the tools create double stranded breaks in target sequences via FokI nuclease activity but differ in their recognition pattern ZFNs have been widely used but researchers have faced issues with in terms of affinity of binding to their target among others The present article effectively explains various aspects of TALEN designing and effectiveness based on parameters such as linker size Although both TALENs and ZFNs have been used for genome editing a comparison of both based on their efficacy was lacking The authors set out to compare the effectiveness of TALENs to generate genomic alterations that have already been attempted with ZFNs The authors also report their results in using TALENs to induce novel mutations in Drosophila genes First the authors generated TALENs for two Drosophila genes ry and y

The target sites overlapped with regions which had previously been targeted by ZFNs this would allow us to directly compare their effectiveness in generating mutations at the target site The results as observed by crossing the resulting flies with known ry and y gene carrying known mutants showed that TALENs were more effective in generating non homologous end joining NHEJ mutations of targets as compared to ZFNs Also TALENs with smaller interdomain linker regions were more effective in mutating the target site as compared to their long linker counterparts Next modifications in the dimerization of ZFNs and TALENs were investigated In this experiment wild type forms of both nucleases were altered to result in ZFNs and TALENs which were functional as heterodimers This was done to see if the resulting heterodimers reduced off target cleavage In case of the ZFNs it was seen that the undesired lethality was abolished by the heterodimers However one of the modified ZFNs with double substitutions showed significantly lower efficacy The same parameter was investigated in TALENs even though off target effects have not been observed in them To allow for unbiased comparison the same targets as ZFN heterodimers were investigated with TALENs showing better results To summarize single and double substitutions in ZFNs reduced toxicity caused by their wild type counterparts but resulted in a reduction of efficacy s compared to the wild type TALENs scored better than ZFNs as such toxicity has not been observed and TALENs with double substitutions scored most effective of all altered nucleases

The authors then went on to assess the type of mutations caused by ZFNs and TALENs Although both nucleases use FokI to generate double stranded breaks the resulting mutations repaired by NHEJ differ ZFNs were observed to produce smaller deletions accompanied by insertion of new bases showing a frequently occurring 4 base insertion TALENs on the other hand showed a larger deletion region and failed to show the recurrent 4 base pair insertions seen in ZFNs TALENs were also seen to favor simple deletions or deletions with insertions at their targets This data becomes essential when scientists decide which tool to use to generate random mutations in their targets In some targets it might be favorable to generate larger deletions accompanied by insertions using TALENs while in others smaller deletions may be preferred to avoid deleting surround sequences achievable by ZFNs The present article showed that two sets of TALENs can be simultaneously introduced into Drosophila thereby targeting two different regions in the genome this was done by introducing the mRNAs in the flies corresponding to the TALENs targeting two different targets So far the study compared ZFNs and TALENs with respect to NHEJ mediated repair following cleavage Next the authors delivered a donor oligonucleotide sequence to mediate homologous repair at the target site TALENs were seen to effectively incorporated the donor sequence and mutate the target with oligos homologous to both strands of the target sequence with results comparable to that of ZFNs Lastly the authors attempted to mutate genes for which successful mutants had not been isolated previously the targets were mutated in most cases except for two of the 17 TALEN pairs designed 

For one of these failed TALENs the authors explain that the long inter linker domain could be the attributing factor For two target genes Psf2 and Sld5 reducing the concentration of injected mRNAs for production of the TALEN in the fly eliminated lethality which was initially observed but resulted in infertile flies The same region targeted in ZFNs showed lethality at early stages of the fly again demonstrating the effectiveness of TALENs over ZFNs In conclusion TALENs were seen to be more effective than ZFNs in terms of having site specific activity with no to low off target activity and resulted in a higher number of desirable mutants TALENs are an important resource that might allow us to generate any mutant desired The present paper was successful in elucidating various aspects of TALENs design and usage such as inter linker domain size and mRNA dosage The various types of possible mutations at the targets were summarized as compared to ZFNs which will allow one to determine the desirable nuclease tool to use for genome editing Based on the current research results it would be interesting to apply the same study to other model organisms such as mice and determine if similar characteristics are seen

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