1. CRISPR CAS9:PRESENTAND FURURE
CRISPR(clusteredregularlyinterspacedshort palindromic repeats)is a family
of DNA sequences found within the genomes of prokaryotic organisms such
as bacteria and archaea These sequences are derived from DNA fragments from
viruses that have previously infected the prokaryote and are used to detect and
destroy DNA from similar viruses during subsequent infections. Cas9 (or
"CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a
guide to recognize and cleave specific strands of DNA that are complementary to
the CRISPR sequence.Cas9enzymes together with CRISPR sequences form the
basis of a technology known as CRISPR-Cas9that can be used to edit genes within
organisms. It is currently the simplest, most versatile and precise method of genetic
manipulation and is therefore causing a buzz in the science world. CRISPR
genome editing technology has been considered as one of the major
biotechnological discoveries of the 21st century. Among other powerful genome
editing options such as ZFN and TALENs the CRISPR systemseems to draw more
attention due to its ease of design as well as its extreme dominance in gene knock-
in and knock-out efficiency.
Currently there has been utilization of the CRISPR-Cas9system in the food and
agricultural industry, particularly in the development of resistant crops with
improved quality and productivity.
CRISPR CAS9 in agriculture
Virus- resistant plant development
Considering that viral infections in crops are believed to decrease global yield by
10-15%, the use of the CRISPR system can provide solutions to overcome these
limitations.
Transgenic geminivirus-resistant plants were previously developed using RNAi
mediated gene silencing.
Resistance to plant disease
The CRISPR-Cas9system was also evaluated for the delivery of mutations in the
TaMLO-A1 and TaMLO-B1 gene of bread wheat to generate transgenic plant
resistant to powdery mildew, which is a common fungal disease caused by fungi.
Rice blast resistance was significantly improved by CRISPR-Cas9sequence-
specific nuclease (C-ERF922) using two sgRNAs targeting the OsERF922gene
CRISPR-mediated mutation in DMR6 of Arabidopsis
thaliana as well as tomato plants with deletions in the SIDMR6-1 gene improved
2. resistance against important pathogens like P. syringae, P. capsici, and
Xanthomonasspp.
Physical and chemical resistance of crop
crops usually have to confront several stress factors during growth, harvest, and
post-processing, which decrease overall yield. CRISPR-Cas9-mediated mutation
targeting ALS1 and ALS2 increased herbicide-resistance in maize,
Nutritional improvement
RNP-delivery of CRISPRCas9machinery resulted in commercial lines with higher
yields without the integration of DNA. These studies were of great commercial
importance since amylopectin potato starch was improved in quality.
Cereal-based products that are high in amylose carry potential health benefits.
Hence, CRISPR-Cas9genome editing machinery became essential to increase
amylose and resistant starch content in cereals such as rice.
Manipulating production of bioactive compounds
Effectively controls the production of bioactive alkaloids in the opium poppy
where it was used to regulate benzyl isoquinoline alkaloids (BIAs) metabolism and
biosynthesis by knocking-out the 4′OMT2 gene.
targeted mutation of specific enzymes in SL biosynthesis can be an efficient
alternative to engineer plant architecture (controlling plant height and tilling) to
reduce crop losses and improve overall yield.
the CRISPR-Cas technology has been instrumental in engineering industrial
production hosts, like the yeast (S. cerevisiae) strains used in brewing.
Safety considerations and global market
CRISPR CAS9 in Animals
Disease resistance/resilience
Production of pigs resistant against infection with the Porcine Reproductive and
Respiratory Syndrome (PRRS)Virus (PRRSV)via genetic knockout of the CD163
receptor.
Cattle that became resistant against an infection with M. bovis with the aid of
DNA nuclease-mediated genetic modifications.
Improved performance
The MSTN (myostatin gene) knockout leads to enhanced formation of skeletal
muscles which could be beneficial for meat production
3. Production of allergen removed or allergen free animal products
Using CRISPR/Cas, the genes encoding for ovalbumin and ovomucoid have been
knocked out in an effort to remove the two major allergenic components from egg
white. This could render eggs digestible for wider range of consumers that could
otherwise not consume chicken eggs.
Knocking out of bovine whey protein B lactoglobulin could render bovine milk as
the major animal-derived protein source in human consumption, acceptable for a
bigger number of consumers that could otherwise not consume milk.
Biomedicine
Genetic knockout of a number of genes, including α1,3-galactosyltransferase
(GGTA1- gene), that encodes a sugar epitope on the surface of porcine cells and
plays a major role in xenotransplantation),
the knockout of genes coding for PPAR-Ɣ (peroxisome proliferator-activated
Receptor Gamma) and LDL (Low density lipoprotein) to produce large animal
models for cardiovascular diseases
to produce a model for genetically induced muscular dystrophy),
APC (Adenomatous-polyposis-coli Protein) to generate a model for certain types of
intestinal cancer and the knockout of the gene coding for von Willebrand factor
(vWF) to create a model for coagulation disorders
Furthermore, pigs with a genetic knockout of the MHC system, which have been
produced using CRISPR/Cas are putative universal organ donors for
xenotransplantation.
Humans
tat and rev-targeting CRISPR/Cas9 lentiviral constructs successfully abolished
regulatory protein expression and function, and inhibited HIV-1 replication in
persistently infected CD4+ T-cell lines as well as latently infected T cells.
Future trends
Other members of the CRISPR/Cas family such as Cfp1 could further change the
field oflivestock genome editing. Thedevelopment ofbaseeditors to change a single
nucleotide within the genome without the need to introduce a double-stranded DNA
4. break and to rely on the inner cell repair mechanisms could also be another step
ahead.
Due to the high degree of physiological similarity with humans, porcine organs are
considered as promising solution to satisfy the growing demand of human organs
for allotransplantation. To achieve this goal and to avoid immune rejection
responses, the porcine genome has to be modified to ensure long-term survival of
porcine organs in patients after xenografting. ZFNs, TALENs and CRISPR/Cas
can now be used to elegantly knockout candidate pig genes or to precisely knock-
in transgenes at specific genomic sites in the porcine genome to producepigs
specifically tailored as organ donors.
CRISPR/Cas seems to show the greatest promise and flexibility for genetic
engineering, sequence requirements within the PAM sequence may constrain some
applications. Therefore, evolution of the Cas9 protein should pave the way towards
PAM independence, and may also provide means to generate an even more
efficient Cas9 endonuclease.
To Knock out the Huntington causing gene is the research ongoing in USA.
The mechanism of dissociation of Cas9 from designed sgRNA
and its consequent recycle are still unclear and need further exploration.
One of the remaining challenges in plant editing is the effectiveness of the
transmission
rate of the mutation to the next generations.
Future of CRISPR Cas9is very promising and with this hope of achieving food
security, nutritional safety, disease resistant plants and animals and many more.
But it should be strictly practiced under law without violating human rights and
animal ethics. It should be guided by the law and under strict supervision of
controlling body.
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