Crispr: Unveiling Nature's Genetic Editor and Its Game-Changing Applications

 CRISPR: From Discovery to Revolutionary Applications

Introduction:

CRISPR-Cas9, the cutting-edge gene-editing technology, has revolutionized the field of genetic engineering in recent years. Its remarkable precision and efficiency have opened up unprecedented possibilities in medicine, agriculture, and environmental solutions. In this blog post, we will delve into the latest research on CRISPR, including groundbreaking advancements in 2023 and ongoing projects that are pushing the boundaries of this transformative technology.

I. The Origins and Mechanism of CRISPR:

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was initially discovered as a bacterial immune system in the 1980s. However, it was in 2012 that Jennifer Doudna and Emmanuelle Charpentier demonstrated the potential of CRISPR-Cas9 as a versatile gene-editing tool. The CRISPR-Cas9 system consists of a guide RNA molecule and the Cas9 protein, which work together to target specific DNA sequences and make precise modifications.

II. Medical Breakthroughs:

a) Treating Genetic Diseases:

In 2023, significant progress has been made in using CRISPR to treat genetic disorders. Clinical trials have shown promising results in correcting genetic mutations responsible for conditions such as sickle cell disease, cystic fibrosis, and muscular dystrophy. CRISPR-based therapies offer the potential for long-lasting and targeted treatments, providing hope for patients with previously untreatable genetic diseases.

b) Precision Cancer Therapies:

CRISPR is also being explored as a potential tool in precision cancer therapies. Researchers are utilizing CRISPR to target and disable specific genes involved in cancer growth, metastasis, and drug resistance. This approach could lead to more effective and personalized treatments with fewer side effects.

c) Viral Disease Interventions:

CRISPR holds promise for combating viral diseases. Ongoing research focuses on using CRISPR to target and disable viral genes, offering potential treatments for infectious diseases such as HIV/AIDS, hepatitis, and influenza. Additionally, CRISPR-based diagnostic tools are being developed for rapid and accurate detection of viral pathogens.

III. Advancements in Agriculture:

CRISPR has tremendous potential to enhance agricultural practices, addressing challenges such as crop yield, nutritional content, and pest resistance.

a) Improved Crop Traits:

Researchers are utilizing CRISPR to develop crops with desirable traits, such as increased drought tolerance, disease resistance, and improved nutritional content. For instance, ongoing projects aim to enhance the nutritional value of staple crops by modifying genes responsible for vitamin and mineral production.

b) Sustainable Agriculture:

CRISPR is being explored to develop environmentally friendly agricultural solutions. Scientists are utilizing CRISPR to engineer plants with enhanced nitrogen fixation capabilities, reducing the need for synthetic fertilizers. Additionally, CRISPR can help combat plant diseases by precisely editing genes to enhance resistance, reducing the reliance on chemical pesticides.

IV. Environmental Solutions:

CRISPR-based interventions have the potential to address pressing environmental challenges.

a) Climate Change Resilience:

Researchers are investigating the use of CRISPR to modify the genes of various organisms, including trees and corals, to enhance their resilience to climate change stressors. This approach aims to develop climate-adapted species that can better withstand rising temperatures, drought, and other environmental pressures.

b) Ecosystem Restoration:

CRISPR offers opportunities for ecosystem restoration by targeting invasive species that disrupt ecological balance. Scientists are exploring methods to edit genes in invasive organisms to suppress their populations and restore native biodiversity. Furthermore, CRISPR may be employed to modify microorganisms capable of degrading pollutants, facilitating environmental cleanup efforts.

V. Ethical Considerations and Regulatory Framework:

As CRISPR continues to advance, it raises ethical considerations and requires a robust regulatory framework.

a) Germline Editing and Ethical Boundaries:

Germline editing, altering the genetic material of embryos or reproductive cells, raises ethical questions regarding the potential for heritable changes. Ongoing discussions are focused on defining the ethical boundaries of germline editing and establishing guidelines for responsible use.

b) Responsible Innovation and Regulation:

To ensure the safe and ethical use of CRISPR, regulatory frameworks are being developed worldwide. These frameworks aim to strike a balance between enabling scientific progress and addressing potential risks associated with human applications, environmental impacts, and unintended consequences.

Conclusion:

CRISPR-Cas9 has revolutionized genetic engineering, enabling precise and efficient gene editing with far-reaching implications. Ongoing research in 2023 continues to expand the applications of CRISPR, from treating genetic diseases and combating cancer to enhancing agricultural practices and addressing environmental challenges. However, it is essential to navigate ethical considerations and establish robust regulations to ensure responsible use and harness the full potential of this remarkable technology. With ongoing advancements and groundbreaking projects, CRISPR remains at the forefront of scientific innovation, holding the promise to reshape our world for the better.

 CRISPR: Transforming Plant Research and Revolutionizing Agriculture

Introduction

In the realm of scientific innovation, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a game-changing tool, enabling precise gene editing and holding immense potential for plant research and agriculture. This revolutionary technology has opened up new avenues for developing crops with improved traits, disease resistance, and enhanced nutritional value. In this blog, we will explore the latest research in plant CRISPR applications and the transformative impact it has on agriculture.

                                                                      Fig.1

Precision Genome Editing: Tailoring Plants to Perfection

CRISPR-Cas9 technology has revolutionized plant research by enabling precise modifications to the plant genome. Scientists can target specific genes responsible for traits such as yield, stress tolerance, and nutritional content, and introduce desired changes. By leveraging CRISPR, researchers have been able to develop plants with enhanced qualities, such as disease resistance in crops like rice, wheat, and maize. This precision genome editing offers immense potential for addressing global challenges, including food security and sustainable agriculture.

Improving Nutritional Content: Biofortification through CRISPR

One of the key areas of focus in plant research is biofortification, enhancing the nutritional value of crops to combat malnutrition and dietary deficiencies. CRISPR has been instrumental in this field by enabling targeted modifications in plant genes responsible for nutrient production. For example, scientists have used CRISPR to enhance the iron and zinc content in staple crops like rice, wheat, and cassava. This breakthrough offers a sustainable solution to address micronutrient deficiencies and improve human health on a global scale.

                                                                           Fig.2

Disease Resistance: Enhancing Plant Immunity

Crop diseases can cause significant losses in agricultural productivity. CRISPR technology has the potential to develop crops with enhanced disease resistance, reducing the reliance on chemical pesticides and promoting sustainable farming practices. Researchers have successfully used CRISPR to confer resistance to devastating plant diseases such as citrus greening in oranges, late blight in potatoes, and bacterial blight in rice. By editing specific genes involved in disease susceptibility, scientists can create crops that are better equipped to withstand pathogen attacks, leading to increased crop yields and reduced environmental impact.

Climate Adaptation: Developing Resilient Crops

Climate change poses a significant threat to global agriculture, with rising temperatures, droughts, and extreme weather events impacting crop productivity. CRISPR technology offers a powerful tool for developing climate-resilient crops. By modifying genes associated with stress responses, such as those involved in drought tolerance or heat resistance, researchers can create plants better suited to withstand changing environmental conditions. This research can contribute to the development of climate-smart agriculture, ensuring food security in the face of a changing climate.

Gene Regulation: Beyond DNA Editing

In addition to precise DNA editing, CRISPR has opened up new possibilities in gene regulation. Researchers are exploring CRISPR-based technologies like CRISPRi (interference) and CRISPRa (activation) to modulate gene expression in plants. This approach allows for fine-tuning of gene activity without making changes to the DNA sequence. By selectively activating or repressing specific genes, scientists can influence traits such as flowering time, fruit ripening, and hormone responses. This innovative use of CRISPR expands the toolkit available for plant researchers and offers exciting prospects for crop improvement.

                                                                        Fig.3

Conclusion

CRISPR technology has ushered in a new era of plant research and agricultural advancement. With its precision genome editing capabilities, CRISPR holds the potential to revolutionize crop breeding, improve nutritional content, enhance disease resistance, and develop climate-resilient varieties. By harnessing the power of CRISPR, scientists and researchers can pave the way for sustainable agriculture, food security, and a healthier future. As we continue to explore the possibilities of this transformative technology, it is crucial to uphold ethical considerations, promote responsible use, and engage in open dialogue to maximize its positive impact on plants, agriculture, and society as a whole.

CRISPR, TALENs mixed to Edit Mitochondrial DNA

CRISPR, TALENs mixed to Edit Mitochondrial DNA 

( CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats )


( TALENs - Transcription activator-like effector nuclease )

Gene editing 

US researchers have constructed up a technique for making exact alters to mitochondrial DNA inner dwelling cells. 

Analysts from the Wide Institute of MIT and Harvard in Massachusetts and the college of Washington college of medication constructed up until some other methodology that consolidates competencies of CRISPR-primarily based base improving with a more pro manner called TALENs, to deal with component ameliorations within the mitochondrial genome. 

'It's a quantum take off forward' expressed the extensive Institute's Professor Vamsi Mootha, one of the creators of the examination, that is distributed in Nature. 'That is the vital time in my vocation that we have had the choice to layout a specific modify in mitochondrial DNA.' 

DNA 

Mitochondria are impartial organelles located in pretty plenty each flexible in the human casing, in which they produce the power to strengthen the portable. Mitochondria comprise their very own special genomes – roundabout components of DNA completely split away

chromosomes within the cell's nucleus. 

While the mitochondrial genome changed into the preliminary section of human DNA to be sequenced, it has developed to be one of the closings to be comfortably altered. 

CRISPR/Cas9 genome editing – which has been splendidly powerful in nuclear DNA accomplishes not in mitochondria in light of the truth that the guide RNA debris can't move the film that encompasses every mitochondrion. Moreover, the DNA restoration additives that retouch the double deserted breaks due to Cas9 are absent in mitochondria. 

Beyond techniques of genome altering, for example, zinc-finger nucleases and TALENs had been ready to reduce mitochondrial DNA anyway should harm it within the method, rendering centered changes incomprehensible. 

The brand new technique saddles a poison determined in small scale living being to trade a cytosine base inside the DNA code to thiamine, hence has the potential to unique a few factor changes in the mitochondrial genome. The revelation of this poison by using utilizing analysts at the University of Washington drove Professor Joseph Mougous to touch Professor David Liu at the extensive Institute, who's perceived for growing base changing (see BioNews 848). 

Collectively, the labs labored to deliver the proofreader in a way that refuted its harmfulness and matched it with a story (interpretation activator-like effector) – a protein guide just like the ones applied in TALENs, dislike CRISPR RNA can cross the mitochondrial movie. 

'A mitochondrial genome editorial supervisor has the long-term time frame potential to be advanced into a beneficial to manipulate mitochondrial-determined disorders, and it has a more instantaneous fee as a tool that researchers can use to higher edition mitochondrial ailments and check out basic inquiries regarding mitochondrial science and hereditary features.

Mitochondrial DNA 

Mitochondria 

Mitochondria include their own genome, which encodes 13 proteins which can be subunits of breath chain homes, just as rRNAs and 22 mitochondrial tRNAs. 

Because of the primary jobs of genes encoded with the aid of the method of mtDNA, the renovation of mitochondrial genome trustworthiness is truly enormous for ordinary cell capacities. 

Mitochondrial DNA is, but, continually below mutational weight because of oxidative stress compelled through radicals produced by making use of oxidative phosphorylation or an awkwardness within the mobile reinforcement protect framework in turning into greater established or disease strategies. 

Damage to mtDNA, as an instance, element modifications or cancellations, provides to or inclines human beings to a collection of human illnesses. Especially, vain mitochondria have been ensnared in various neurodegenerative afflictions inclusive of Parkinson's ailment. 

However the large capacity of mitoTALEN-intervened mtDNA upgrading, extra client inviting, and proficient elective strategies are crucial to overcoming demanding situations in mtDNA change both for rectification of vain mtDNA or for delivering vain mtDNA for you to make mitochondria-associated illness models. 

 REFERENCES 

A bacterial cytidine deaminase poison allows CRISPR-loose mitochondrial base changing 8 July 2020 

The quality altering disclosure may want to component the course extra like a 'sacred purpose': remedy plans for mitochondrial maladies 8 July 2020 

The handiest method to precisely Edit Mitochondrial DNA 

Howard Hughes Medical Institute eight July 2020 

Mitochondrial genome improving receives genuine 8 July 2020 

New technique to regulate the cellular's 'force to be reckoned with' DNA might need to help view dissemination of hereditary diseases

Researchers make exciting first-class alters to mitochondrial DNA for the essential 8 July 2020 

The forces to be reckoned with inner cells had been finely altered for the essential time

Genome editing technology

What is Genome Editing? 

•Genome modifying, or genome engineering, or gene modifying, may be a quite hereditary designing where DNA is embedded, erased, changed or supplanted within the genome of a living organic structure.

•Unlike early hereditary building methods that arbitrarily embeds hereditary material into a bunch genome, genome altering focuses on the additions to site specific locations.

Genome editiors 

As of 2015 four groups of built nucleases were utilized: meganucleases, zinc finger nucleases (ZFNs), interpretation activator-like effector-based nucleases (TALEN), and therefore the bunched routinely interspaced short palindromic rehashes (CRISPR/Cas9) framework.

•Nine genome editors were accessible starting at 2017. 

•All three significant classes of those compounds—zinc finger nucleases (ZFNs), interpretation activator-like effector nucleases (TALENs) and designed meganucleases—were chosen naturally Methods because the 2011 Method of the Year.

•The CRISPR-Cas9 framework was chosen by Science as 2015 Breakthrough of the Year. 

• The nucleases create specific double-strand breaks (DSBs) at desired locations within the genome and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end- joining (NHEJ).

 

Utilizations:  

Gene knock out

Gene tagging 

Unique mutation (insertion/deletion study) 

Gene knock in 

Promoter have a glance at

Strategies for plant genome control 

Classical breeding 

Transgenic technique 

Targeted genome enhancing

Meganucleases 

•Meganucleases, found within the late 1980s, are catalysts within the endonuclease family which are portrayed by their ability to perceive and cut huge DNA successions (from 14 to 40 base sets).

•The maximum widespread and excellent acknowledged meganucleases are the proteins within the LAGLIDADG own family, which owe their call to a conserved amino acid collection.

•Meganucleases have the advantage of inflicting less toxicity in cells than methods inclusive of Zinc finger nuclease (ZFN), in all likelihood because of extra stringent DNA series recognition.

•One fundamental disadvantage is the construction of sequence-specific enzymes for all possible sequences is steeply-priced and time consuming, as one is not benefiting from combinatorial possibilities that methods including ZFNs and TALEN- based totally fusions utilize. 

 

Crop in which it is used 

Maize : Herbicide obstruction ( Gao et al, 2010 ) 

Cotton : Herbicide obstruction and and bug opposition ( D'Halluin et al., 2013 ) 

Impediment 

Hard to regulate the DNA restricting site 

Little recognition site

What is ZFN innovation?  

Zinc fingers were first found within the African pawed amphibian (Xenopus laevis) in 1985.

A class of designed DNA-restricting proteins. 

Encourage targated altering of the genome by making double strand breaks inside the DNA at specific places. 

Double strand breaks are important for site-explicit mutagenesis. 

Invigorate the cell’s natural DNA repair methods i.e, HR and NHEJ  

Generate exactly targeted genomic editing leading to cell strains with targated gene deletions, integrations, or modifications.

 

What are zinc finger nuclease 

Extraordinarily particular genomic scissor 

Consists of two practical domains 

• A DNA – binding domain 

•  A DNA- cleaving domain accommodates of nuclease area of FoK I ( FokI could be a obviously going down type IIS restrict enzyme and is found in Flavobacterium okeanokoites.

Crop in which it turned into used

Maize : Herbicide resistance ( Shukla et al., 2009 ) 

Soybean : Physiological quality ( Curtin et al., 2011 ) 

Tomato : Towards TYLCV  ( Takenaka et al., 2007 ) 

Impediment  

Off objective impact  

Development is lumbering and tedious 

Uses of ZFN 

•Repairing mutations 

•Insertion of gene or DNA piece at explicit site 

•Repair or supplant distorted genes 

•Disabiling an allele 

•Allele altering 

•Applications in clinical segment 

• a) Gene treatment 

• b)Treatment of HIV

TALENs ( Transcription activator-like effector nucleases )

Transcription activator-like effector nucleases TALENs are the restriction nuclease engineered to scale back precise sequences of DNA . They're made by means of fusing: DNA-binding domain (TAL effector) DNA-cleavage area ( the catalytic domain of RE FoK I).

TALENs may be designed to tie any ideal DNA succession to chop at explicit areas in DNA. First time revealed by Ulla Bonas in Xanthomonas oryzae (1989).

 

TALEN constructs are utilized in a very comparable manner to designed zinc finger nucleases and have three blessings in focused mutagenesis

1. DNA binding specificity is higher.

2. Off-target impacts are lower, and 

3. 3. Development of DNA-binding domain names is less complicated.

Primarily based on the most theoretical distance between DNA binding and nuclease hobby, TALEN methods lead to the best precision.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)

 

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are hereditary components that microorganisms use as a kind of gained insusceptibility to secure against infections. They comprise of short arrangements that begin from viral genomes and are fused into the bacterial genome.

Cas (CRISPR associated proteins) manner these sequences and reduce matching viral DNA sequences.

With the help of introducing plasmids containing Cas genes and explicitly developed CRISPRs into eukaryotic cells, the eukaryotic genome are often reduce at any desired position. some organizations, including Cellectis and Editas are attempting to adapt the CRISPR technique while creating gene explicit treatments.

Parts of CRISPR 

Proto spacer adjacent motif (PAM) could be a DNA arrangement promptly following the DNA succession focused by the Cas9 nuclease within the CRISPR bacterial versatile resistant framework.

PAM may be a component of the invading virus or plasmid, however isn't a thing of the bacterial CRISPR locus. Cas9 won't efficiently bind to or cleave the target DNA sequence if it's not followed by way of the PAM sequence.

CRISPR RNA (crRNA) may be a trans-encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts.

Trans activating crRNA (tracr RNA ) is formed of of a more drawn out stretch of bases that are consistent and provides the "stem circle" structure limited by the CRISPR nuclease.

 At the point when these RNA parts hybridize they structure a guide RNA which "programmably" targets CRISPR nucleases to DNA arrangements relying upon the complementarity of the crRNA and therefore the presence of various DNA capabilities (PAM collection identified by the nuclease).

 

Examples of plants changed with CRISPR innovation 

Corn : Targeted mutagenesis ( Liang et al. 2014 ) 

Rice : Targeted mutagenesis ( Belhaj et al. 2013 ) 

Sorghum : Targeted gene alteration ( Jiang et al. 2013b ) 

Sweet orange : Targeted genome altering ( Jia and Wang 2014 ) 

Tobacco : Targeted mutagenesis ( Belhaj et al. 2013 ) 

Utility in Agriculture

1. Can be utilized to make serious extent of hereditary inconstancy at exact locus in the genome of the crop plants.

2. Capacity device for multiplexed converse and forward hereditary study. 

3. Exact transgene joining at explicit loci.  

4. Developing biotic and abiotic safe attributes in crop plants.  

5. Potential tool for growing virus resistant crop types.

6. Can be utilized to eliminate  undesirable species like herbicide resistant weeds, insect pest.

7. Ability device for improving polyploid crops like potato and wheat.

References

Jasin M (June 1996). "Genetic manipulation of genomes with rare-cutting endonucleases". Trends in Genetics. 12 (6): 224–8. doi:10.1016/0168-9525(96)10019-6.

 Science News Staff (17 December 2015). "Breakthrough of the Year: CRISPR makes the cut.

Tan WS, Carlson DF, Walton MW, Fahrenkrug SC, Hackett PB (2012). Precision editing of large animal genomes. Advances in Genetics. 80. pp. 37–97. doi:10.1016/B978-0-12-404742-6.00002-8.

Cheong, Kang Hao; Koh, Jin Ming; Jones, Michael C. (2019). "Black Swans of CRISPR: Stochasticity and Complexity of Genetic Regulation". BioEssays. 0 (7): 1900032. doi:10.1002/bies.201900032.