CRISPR/CAS9 AND PLANTS

Technical Writing and

Presentation
 CRISPR/CAS9 AND PLANTS

Review
CRISPR/Cas9 System: Application in some crops
Tuyet Anh (20180303), Nguyen Diu (20180313), Thuy Hang (20180327), Vu Phuong (20180363)
Genome editing (GE) has marked the development biological research as it can be used to edit the
genomes of living organisms. There are manny tools that have been used and the tool is widely used in
GE due to ít high efficiency, ease of use, accuracy is clustered regularly interspaced short palindromic re-
peats (CRISPR)/Cas9 system. It can be used to add desirable and remove undesirable alleles
simultaneously in a single event. Here, we discuss the applications of CRISPR/Cas9 within several crops,
comparisons with other methods, mechanisms, limitations, and future possibil-ities.

Genome Editing
Genome editing is a technique that allows scientists to work on gens by
inserting/deleting or replacing bases in the original sequence. Since 2002 and 2011,
ZFNs and TALENs have usual used. ZFNs stands for technical use of zinc finger
nucleases, which are targetable DNA cleavage proteins used to cut DNA sequences
at any site. Transcriptional activator-like effector nucleases – TALENs induce
double-stranded breaks in target sequences , which trigger DNA damage response
pathways, leading to genome modification. The above two techniques have many
limitations, so since 2013, attention has turned to CRISPR/Cas 9. CRISPR-Cas9 is
a genome editing tool that is make a big buzz in the science world. It is faster,
cheaper price and more accurate than previous techniques of editing DNA and has
a wide range of potential applications. Here, we learn about the applications of
CRISPR/Cas9 in crop plants.
The Mechanism of the CRISPR/Cas9 System
The CRISPR/Cas9 system consists of two key molecules that introduce a change
into the DNA. There are: an enzyme called Cas9 and a piece of RNA called guide
RNA (gRNA).
o gRNA finds the spots and guide the Cas9 to a target
o Cas9 cuts the target DNA
o The cut is repaired
 CRISPR/CAS9 AND PLANTS

To describe the operation of the CRISPR/Cas9, we observe Figure 1

Figure 1: Diagram showing
how the CRISPR-Cas9
editing tool works. Image
credit: Genome Research
Limited.

[CITATION Yourgenome \l 1033 ]

The Beneficial Features and Limitations of the CRISPR/Cas9

System
CRISPR/Cas is a technique that can overcome the disadvantages of previous
methods, it is easier to use and cheaper and much more eficient. However, it also
has come limitations such as:
o CRISPR/Cas9 can introduce multiple random off-target mutations in the
genome [ CITATION Zang2015 \l 1033 ]. However, new CRISPR/Cas varia[ CITATION
Manghwar \l 1033 ]nts have improved editing efficiency of target bases in the
 CRISPR/CAS9 AND PLANTS

sequence of interest by recognizing different PAMs [ CITATION KaiHua2019 \l 1033

].
o Difficulties exist for the commercialization of transgenic crops expressing
CRISPR/Cas9 in various countries, primarily because of the development
costs and constraints imposed by regulatory systems for the field release of
genetically modified organisms [ CITATION Manghwar \l 1033 ].
This technique is widely applied in research, medicine… and especially
agriculture.
Transgenic steps move CRISPR/Cas9 from gene selection to plant
analysis
Analytical materials include: rice[ CITATION Macovei2018 \l 1033 ], cotton[ CITATION Qin19 \l
1033 ][ CITATION LiJ16 \l 1033 ], maize [ CITATION Lee18 \l 1033 ] , soybean[ CITATION LiZ15 \l
1033 ], tomato [ CITATION alA \l 1033 ], potato [ CITATION Enc19 \l 1033 ], cucumber[ CITATION
Cha16 \l 1033 ].

Conduct [ CITATION Manghwar \l 1033 ]:

1) Selection of the target gene.
2) Designing the single-guide RNA (sgRNA) for the target gene.
3) Vector construction.
4) Genetic transformation via Agrobacterium/ribonucleoprotein (RNP) for the
delivery of CRISPR/Cas9.
5) Tissue culture (callus induction).
6) Plant regeneration from CRISPR/Cas9mutated tissues.
7) Generation of T0 CRISPR/Cas9-mutated transgenic plants.
8) Screening of transgenic plants by PCR.
9) Detection of on- and offtarget efficiency of CRISPR/Cas9-mutated plants by
T7E1.
10) Detection of on- and off-target efficiency by Sanger sequencing.
11) Different methods to detect on- and off-target efficiency.
12) Self-pollination of To transgenic plants for generation of homozygous T1 plants.
 CRISPR/CAS9 AND PLANTS

13) CRISPR/Cas9-mutated To seeds.

14) Generation of transgene-free T1 progeny.
15) Phenotypic analysis of T1 plants and other analysis. Abbreviations: Cas9,
CRISPR-associated nuclease 9; CRISPR, clustered regularly interspaced short
palindromic repeat; crRNA, CRISPR RNA; tracrRNA, trans-activating CRISPR
RNA.

Figure 2: Pipeline of CRISPR/Cas9 Genetic Transformation of Genes from Gene

Selection to Plant Analysis.
Results and Discussion
 CRISPR/CAS9 AND PLANTS

The data table 1 below is some specific examples about plants that have adopted
the gene editing technique Crispr/Cas 9:
Table1: The applications of the CRISPR/Cas9 system in major crops
 CRISPR/CAS9 AND PLANTS

The above table is confirmed that Crispr/Cas 9 technology has been successfully
applied and tested on many diverse plant species. For different plants, the gene
editing efficiency is also different, varying from 46 to 100%. This technique is the
most effective when editing gene SIIAA9 of tomato (100%) and gene GhMYB 25-
like of cotton (98,8 – 100%). Conversely, it is less efficient when applied to gene
GhCLA1, GhVP of upland cotton (47,6 – 81,8%); gene TMS5 of rice (46,2 –
88,2%). However, the efficiency obtained from this technology is still higher than
that of other gene editing techniques. The most common method of cas 9 system
delivery used is Agrobacterium – mediated transformation method to insert,
remove, replace nucleotides or cause point mutations, allele mutations of host
genome to help trait improvement in crops. Gene editing by Crispr/Cas 9 offers
many benefits to plants such as the next lives were observed to be stronger and
provie a greater yield compared with control variety or resistant against
sulfonylurea herbicides (rice, cotton, maize); morphological changes in seedless
fruit and leaf shape (tomato); reduction of amylose content in starch (potato);
enhance tolerance against the infection of papaya ring spot mosaic virus
(cucumber). Besides, there are many other traits that have been changed according
to human wishes.
The development of this technique for plants creates mutations in the plants, and
now the question is whether these products are considered GMO? In 2016, an
American research group was granted a patent and allowed to bring Crispr/cas9
products to the market without being GMO[ CITATION Wal16 \l 1033 ]. For Europe, this
issue is not yet officially clear.
 CRISPR/CAS9 AND PLANTS

To overcome the limitations of CRISPR/Cas9, scientists have created many new

Crispr/Cas such as: xCas9 and SpCas9-NG [ CITATION Hua19 \l 1033 ], Cas13, Cas12a
[ CITATION Moo18 \l 1033 ], [ CITATION Zet15 \l 1033 ]...... and got positive results.

Concluding Remarks and Future Perspectives

CRISPR/Cas9 is one of the most interested gene editing techniques today, It
appears to mark a major turning point in the biological research world. Although
there are still some limitations and not really widely used today, scientists have
now improved to be able to edit the genes of humans and plants.

References
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Hua, K . et al. 2019. Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM
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 CRISPR/CAS9 AND PLANTS

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[ CITATION Cha16 \l 1033 ]