Abstract
Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.
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Acknowledgements
Authors acknowledge Central University of Kerala for granting the facility for the study and the funding support from Kerala State Council for Science, Technology and Environment (KSCSTE).
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The research was supported by funds from Kerala State Council for Science, Technology and Environment (KSCSTE) (Reference number 007/FSHP-LSS/2013/KSCSTE).
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JMS have conceived the idea of writing this manuscript and contributed much to design the content. She proposed the strategy to control the off-target effect of CRISPR/Cas9 complex. PPA has performed the literature search and contributed to writing this manuscript. She proposed the strategy for improving efficiency of homologous recombination based targeted knock out. Both the authors contributed to final review and acceptance of the manuscript.
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Amritha, P.P., Shah, J.M. Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies. Mol Genet Genomics 296, 485–500 (2021). https://doi.org/10.1007/s00438-021-01769-y
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DOI: https://doi.org/10.1007/s00438-021-01769-y