This document provides an overview of CRISPR-Cas9 gene editing technology and its applications in food editing. It explains that CRISPR-Cas9 utilizes guide RNA and Cas9 nuclease to precisely target and edit DNA sequences. The document discusses how CRISPR-Cas9 is being used to improve crop traits like yield, nutrition, and disease resistance in tomatoes, rice, wheat, and other plants. While promising for agriculture, the document notes there are still controversies around off-target effects and safety that require further study before wide application of CRISPR gene editing in food.
3. To understand CRISPR we should go back to 1987
When Japanese scientists studying E. coli bacteria first came
across some unusual repeating sequences in the organism’s DNA,
which was unknown. Over time, other researchers found similar
clusters in the DNA of other bacteria (and archaea). They gave
these sequences a name: Clustered Regularly Interspaced Short
Palindromic Repeats — or CRISPR.
Yet the function of these CRISPR sequences was mostly a mystery until
2007.
4. What happened in 2007?
- Streptococcus.
- Adaptive immune system.
- Assaulted from viruses.
- Produce enzymes.
- Scoop up remains of viral genes (either RNA/ or DNA).
- Cut it into tiny bits.
- Store those fragments in CRISPR spaces in the bacterium’s own genome.
CRISPR spaces act a gallery for viruses
5. What is CRISPR-Cas9 ?
ü Clustered Regularly Interspaced Short Palindromic Repeats.
ü Precisely manipulate virtually any genomic sequence specified by a short stretch of
guide RNA.
ü Many bacteria and all archaea have evolved sophisticated RNA-guided adaptive
immune systems encoded by CRISPR loci and the accompanying CRISPR-associated
(Cas) genes to provide acquired immunity against bacteriophage infection.
6. CRISPR-Cas system relies on two main
components: a guide RNA (gRNA) and
CRISPR-associated (Cas) nuclease.
7. Cas9
Nuclease has ability to cleave DNA. There are several versions of Cas nucleases isolated
from different bacteria. The most commonly used one is the Cas9 nuclease
from Streptococcus pyogenes.
gRNA
Guide RNA is a specific RNA sequence that recognizes the target DNA region of interest
and directs the Cas nuclease for editing. It is made up of two parts: crispr RNA (crRNA),
a 17-20 nucleotide sequence complementary to the target DNA, and a -trans-activating-
RNA(tracrRNA), which serves as a binding scaffold for the Cas nuclease.
Components of CRISPR-Cas9
8. Protospacer Adjacent Motif (PAM) is a short DNA sequence (2-6 bp) and a
component of the invading NA that follows the DNA region targeted for
cleavage by the CRISPR system, that varies depending on the bacterial
species of the Cas9 gene.
The PAM of the most commonly used SpCas9 (Cas9) from Streptococcus
pyogenes) is ‘NGG’
HOW can the bacteria distinguish between itself and the viral/foreign gene?
PAM
9. - RNA reads genetic information in DNA.
- gRNA shepherds Cas9 to the precise spot on DNA where a cut is called for.
- Cas9 then locks onto the double-stranded DNA and unzips it.
- This allows gRNA to pair up with the region of the DNA it has targeted.
- Cas9 snips the DNA at this spot, which creates a break in both strands of the DNA
molecule. (DSB)
- the DSB triggers DNA repair.
- Fixing the break might disable a gene. Alternatively, this repair might fix a mistake or
even insert a new gene.
How It Works?
11. The two repair pathways of chromosomal DSBs
1. Non-homologous end joining (NHEJ), directs ligation of two break ends with little or no sequence
homology required, resulting in small insertions or deletions (indels). Susceptible to frequent
mutation errors due to nucleotide (indels).
2. Homology-directed repair (HDR), repairs DSB according to a DNA template with homology
sequence, resulting in precise editing when supplemented with ds- or ss-DNA donors. Less errors
or chances of mutations if the DNA template used during repair is identical to the original
undamaged DNA sequence.
In case of CRISPR, the population of transfected cells will contain a combination of NHEJ-repaired
and HDR-repaired alleles. HDR-edited DNA is much more desirable to ensure controlled modifications.
12.
13. Current Applications of CRISPR-Cas9
1. CRISPR tomatoes: groundcherry, A productivity improvement in the genes that control the size of the plant, the
size of the tomato, how many fruits are produced, and the plant architecture. Lemmon et al.
2. CRISPR mushrooms: stop them from browning, A small deletion targeting the polyphenol oxidase (PPO)
family of genes to prevent browning. A successful knockout of one of six PPO genes reduced browning activity by 30%.
Yinong Yang
3. CRISPR rice: improving the yield, Mutations in a family of genes involved in sensing abscisic acid, a phytochrome
that affects plant growth and stress responses. A subset of mutations in specific groups of genes resulted in a 25-31%
increased grain yield. Miao et al.
4. CRISPR wheat: removing the gluten, an 85% reduction in immunoreactivity, and a successful mutation of
35/45 genes of the wild species of wheat to produce a transgene-free, low-gluten species of wheat. Sánchez-Léon et al.
14. 5. Coffee, Natural decaffeination to
stop the expense of removing
caffeine from coffee beans. Awaiting
global regulatory approval.
15. Weather resistance
Drought resistance
Pest resistance
End global use of pesticides
Improve produce longevity and nutrition
Produce with better nutritional value
Helping those with allergies and sensitivities enjoy food
16. Gene Editing vs Genetically Modified Organisms?
CRISPR GMOs
Highly accurate Less accurate
Native DNA DNA is exotic
Cost effective Expensive
17. CRISPR tomorrow
Interest in the field of CRISPR-Cas9 has rapidly increased in recent years. On
the other hand, there are numerous controversies related to CRISPR, including
off-target effects, the immunogenicity of Cas9 nucleases and carcinogenic
effects of CRISPR components, which require exhaustive analysis and scientific
explanations.
18. References:
- Ansari, W. A., Chandanshive, S. U., Bhatt, V., Nadaf, A. B., Vats, S., Katara, J. L., Sonah, H., & Deshmukh, R. (2020).
Genome Editing in Cereals: Approaches, Applications and Challenges. International journal of molecular sciences, 21(11),
4040.
- Ding, W., Zhang, Y., & Shi, S. (2020). Development and Application of CRISPR/Cas in Microbial Biotechnology. Frontiers
in bioengineering and biotechnology, 8, 711.
- Tofazzal, I. (2019). CRISPR-Cas technology in modifying food crops. CAB Rev. 14, No. 050.
- Redman, M., King, A., Watson, C., & King, D. (2016). What is CRISPR/Cas9?. Archives of disease in childhood. Education
and practice edition, 101(4), 213–215.
- Amanda, M. (2019). CRISPR in Agriculture: An Era of Food Evolution. Synthego; https://www.synthego.com/blog/crispr-
agriculture-foods.
- Anders C, Niewoehner O, et al. (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9
endonuclease. Nature, 513(7519):569–573.
- Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology. 31(9):827–
832.
- M. Jasin and R. Rothstein. (2013) “Repair of Strand Breaks by Homologous Recombination,” Cold Spring Harb Perspect
Biol.
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Ghaida Alrumaizan
438202218
Dr. Hind Alshuwaiman
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