Plant Genome Editing by CRISPR-Cas System
Plant Genome Editing by CRISPR-Cas System
Introduction:
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages and provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas(CRISPR-associated) proteins recognize and cut foreign pathogenic DNA.
Genome editing is a way of making specific changes to the DNA of a cell or organism. An enzyme cuts the DNA at a specific sequence, and when this is repaired by the cell a change or 'edit' is made to the sequence.Genome editing in plants has been made much easier with the recently developed CRISPR/Cas9 system because of its simplicity and versatility.
History:
CRISPR: An Adaptive Immune System.CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences were initially discovered in the E. coli genome in 1987, but their function as a safeguard against bacteriophages was not elucidated until 2007.Protospacers are separated by short palindromic repeat sequences.
Three different Types of CRISPR-Cas system;the CRISPR/Cas systems have been classified into three distinct types: A) type I,B) type II andC) type III. While types I and III are found in both bacteria and archaea, type II is unique to only bacteria.
Genome-editing technologies:
Three technologies have been developed as major genome-editing technologies.
At first, zinc finger nuclease (ZFN) has been reported as an engineered nuclease.
Secondary, transcription activator-like effector nuclease (TALEN) has appeared as a more flexible engineered nuclease.
Advance Genome-editing technologie:
Finally, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) has been developed as a more simple and flexible engineered nuclease.
ZFNs and TALENs consist of a sequence-specific DNA binding module and a FokI nuclease domain, it is expensive and difficult to design active nucleases . By contrast, the CRISPR/Cas9 system is inexpensive, and the experimental design is easy. The CRISPR/Cas9 system emerged as a genome-editing tool in 2012 .
The simplicity, ease, and high efficiency of the CRISPR/Cas9 system have facilitated its development into the most widely applied genome-editing tool.
Method of genome editing by
CRISPR_Cas system:
CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system– binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA.
Steps of genome editing by CRISPR_Cas
system are:
(1)Select genomic target . (2)Design sgRNA (small guide RNA). (3).Assemble Cas/sgRNA Construct .(4)Deliver to plant .(5)Regenerate and screen transgenic plant for gene editing events
Genome editing using CRISPR/Cas9 in plants: an overview
The CRISPR/Cas9 system has been successfully applied in various plant species. These include not only model plants, such as Arabidopsis, but also crops, such as rice, tobacco, sorghum, wheat, maize, soybean, tomato, potato, poplar, apple and banana. Calli, leaf discs, protoplasts and flowers have been used as a plant material.
Significance of plant genome editing by Crispr sac system:
Advantages of CRISPR Genome Engineering, the most important advantages of CRISPR/Cas9 over other genome editing technologies is its simplicity and efficiency.
Applications of Genome Editing in Crop
Improvement Genome editing with CRISPR-Cas9 is Amenable to edit any gene in any plant species. Because of its simplicity, efficiency, low cost, and the possibility to target multiple genes, it allows faster genetic modification than other techniques.
It also can be used to genetically modify plants that were previously neglected. The potential that this represents for crop breeding and the development of sustainable agriculture is incommensurable.
Impressive genetic modifications have been achieved with CRISPR-Cas9 to enhance metabolic pathways, tolerance to biotic (fungal, bacterial or viral pathogens), or abiotic stresses (cold, drought, salt), improve nutritional content, increase yield and grain quality, obtain haploid seeds, herbicide resistance, and others.
Notable cases include thermosensitive genic male sterility in maize and wheat, improved nutritional properties in sorghum and wheat tolerance or resistance to pathogens ,and resistance to herbicides.
In potato CRISPR-Cas9 was used to knockout the gene encoding granule-bound starch synthase (GBSS) in one round of transfection resulting in the development of potato plants that produce amylopectin starch, a highly desirable commercial trait .
In cucumber CRISPR-Cas9 system was used to inactivate the eukaryotic translation initiation factor gene elF4E. The resulting non-transgenic homozygotic mutant plants were immune to Cucumber vein yellowing virus (Genus Ipomovirus) and resistant to the potyviruses Zucchini yellow mosaic virus and Papaya ringspot mosaic virus.
Engineering genetic resistance to viruses and other pathogens has immense potential to manage diseases for which no natural resistance has been detected, such as maize lethal necrosis disease and tomato brown rugose fruit virus.
Production of plants with improved traits
drives the current reliance of agriculture and various industries on plant resources.
Traditionally the plant breeding has been
done by crossing and selection. However, the traditional breeding methods are labor- and time-intensive. Genome editing allows targeting and modifying specific DNA
sequences.
Limitations of the CRISPR/Cas9 System:
The molecular mechanism exploited to insert DNA fragments (e.g. cDNAs) is mediated by DNA repair machinery activated by the double strand break introduced by Cas9. Since the scope of the DNA repair system is not to integrate DNA fragments in the genome, targeted alleles often carry additional modifications, such as deletions, partial or multiple integrations of the targeting vector, and even duplications.
Secondary unwanted mutational events at the target locus plague standard ES cell based projects as well, and researchers have learned how to avoid generating mice carrying passenger mutations.
When performing the CRISPR/Cas9 procedure directly on embryos, on the other hand, it is impossible to select for the desired event, greatly limiting the possibility to identify the desired allele. Moreover, the mosaicism observed in founder mice generated using the
CRISPR/Cas9 approach makes the identification of unwanted genomic modifications at the target site very challenging.
References:
Barrangou R, van der Oost J (2013). CRISPR-Cas Systems : RNA-mediated Adaptive Immunity in Bacteria and Archaea. Heidelberg: Springer. p. 6. ISBN 978-3-642-34656-9.
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (August 2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology. 151 (Pt 8): 2551–2561. doi:10.1099/mic.0.28048-0. PMID 16079334.
Comments
Post a Comment