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In-depth inventory: An article takes you to understand gene editing technology

 Last Update: 2022-02-20
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Gene editing technology can sequence the DNA of affected families to provide detailed information on the mutations that cause each disease, as well as the correlation between specific genetic changes (genotypes) and disease severity
.

Genetic therapy is then delivered through changes and modifications of DNA sequences that disrupt the function of toxic or inhibitory genes or restore the function of essential genes

The current gene editing technologies include ZFNs (zinc-finger nucleases) technology, TALENs (transcription activator-like effector nucleases) technology, CRISPR/Cas9 technology, BE (base editing) and PE (prime editing) technology
.

ZFNs, TALENs and CRISPR/Cas9 technologies are used to repair broken DNA by means of non-homologous end joining or homology-directed recombination repair
.

ZFNs, TALENs and CRISPR/Cas9 technology

BE can replace a specific base pair at a specific site with another specified base pair
.

PE can integrate a piece of single-stranded DNA into the genome through the cell's self-repair mechanism, and the original sequence is removed through the cell's repair mechanism
.

Figure 1.
How different gene editing technologies work

Current gene editing technologies treat patients through somatic cell modifications
.

These treatments are designed to affect only the individual receiving the treatment and not their offspring

Current gene editing technologies treat patients through somatic cell modifications

Figure 2.
Germ cell editing

01.
ZFNs technology

ZFNs (zinc-finger nucleases) are artificial nucleases composed of a zinc finger protein domain that determines their specificity and a Fok I nuclease domain that cleaves DNA
.

Figure 3.
a is a schematic diagram of the interaction between ZFNs and DNA, b is a schematic diagram of the triple zinc finger structure pairing DNA

Zinc finger structure is a motif used by the DNA-binding domain of zinc-coordinated DNA-binding domain-like transcription factors
.

There are more than 700 proteins in the human genome that contain at least 4,000 such domains, accounting for about 2% of human genes

Figure 4.
C2H2 zinc finger pdb (where the red balls are zinc ions)

One of the most common zinc fingers is the C2H2 type zinc finger, which is composed of two cysteines and two histidines coordinated with the zinc ion in the middle to stabilize the entire structure
.

Figure 5.
The pdb structure of the second zinc finger of human transcription factor sp1, the gray ball in the middle of the picture is zinc ion

After the structure is stabilized, the zinc finger specifically recognizes the bases of DNA through three positions -1, 3, and 6 of its alpha helix
.

Figure 6.
The pdb structure of the third zinc finger of human transcription factor sp1

As shown in the figure above, R at position -1 and R at position 6 specifically recognize G, and E at position 3 specifically recognizes
C.

Fok I, a member of the Flavobacterium okeanokoites restriction modification system, recognizes a dsDNA non-palindromic sequence 5'-GGATG-3'-5'-CATCC-3'
.

After recognition, it cleaves 9 nt downstream of GGATG and 13 nt downstream of CATCC to form a sticky end, which means it has two domains - a DNA recognition domain and an endonuclease domain
.

Figure 7.
Pdb map of Fok1 protein

FOk1 must be dimerized to be active, and the formation of this dimer requires magnesium ion assistance
.

Figure 8.
Schematic diagram of Fok1 protein dimer, D1, D2, D3 represent the three domains of magenta, green and white, and the blue domain is the endonucleolytic domain

First, the Fok I monomer binds to DNA at its recognition site
.

In the presence of Mg2+, the cleavage domain separates from the recognition domain

Figure 9.
Schematic diagram of Fok1 cleavage mechanism

In 1993, Chandrasegaran and Jin Yangjun et al.
connected three zinc finger domains with the hydrolysis domain of one Fok I nuclease to construct the first zinc finger protein nuclease
.

It recognizes DNA sequences through zinc finger domains and performs site-directed cleavage of DNA through dimerization of Fok1

Figure 10.
Schematic diagram of the specific recognition structure of ZNFs and DNA

02.
TALENs technology

The core of TALENs (transcription activator-like effector nucleases) technology is the TALE protein
.

The TALE protein was originally discovered in Xanthomonas, the major causative agent of bacterial leaf spot in peppers and tomatoes

The core of TALENs (transcription activator-like effector nucleases) technology is the TALE protein

Figure 11.
Schematic diagram of the TALE protein structure
.

The red area in the figure represents the tandem repeat sequence, the yellow area represents the nuclear localization signal, and the green area represents the acidic transcriptional activation domain
.
The figure shows the first sequence of the tandem repeat, in which amino acids 12 and 13 are variable amino acids

Tandem repeats generally consist of 34 amino acid repeat units and are highly conserved
.
Among them, the amino acids at positions 12 and 13 are highly variable and are variable amino acids (RVDs).
RVDs can recognize four different bases
.
Each repeat sequence identifies one nucleotide, as shown in Figure 2
.
The first repeat at the C-terminus has only the first twenty amino acids identical to other repeat units, which is called semi-repetition
.

Figure 12.
Schematic diagram of the corresponding bases on DNA recognized by repeat sequences in the TALE protein.
The first row represents each sequence in the tandem repeat sequence, represented by the abbreviation of variable amino acids; the second row represents the base corresponding to each sequence

By trying different combinations of RVD amino acids, the following code table was obtained

Figure 13.
The code table corresponding to RVDs and nucleotides in the TALE protein repeat sequence.
The level of the base letter in the figure represents the frequency of pairing of the base with the corresponding variable amino acid sequence, and the numbers in the table represent the base and the corresponding variable amino acid sequence.
Frequency of corresponding variable amino acid sequence pairings

Because TALE has the ability to specifically recognize DNA sequences, similar to zinc finger protein nucleases (ZFNs)
.
Therefore, a new DNA editing tool, called TALENs, can be constructed by coupling the specific recognition region of TALE protein with FokⅠ nuclease
.
Since FokI nucleases require dimerization to function, TALENs also need to act in pairs to cleave target DNA sites
.
At the same time, TALENs technology also has problems such as off-target phenomenon, expensive and time-consuming, and may cause the body's immune response
.

03.
Base editing technology

Base editing (BE) technology offers the possibility of converting single bases without introducing DNA double-strand breaks and exogenous DNA templates
.
In 2016, David R.
Liu's team first reported the CBE system composed of cytosine deaminase and nCas9, which can realize the conversion of CT or GA
.

In 2017, David R.
Liu's team developed an ABE system composed of adenine deaminase and nCas9, which can realize the conversion of AG or TC
.
The BE system is mainly composed of dCas9, sgDNA and deaminase.
dCas9 is an inactive Cas9 enzyme, which has no catalytic activity, but can bind to the target DNA, so that the sgDNA (single guide DNA) is combined with the complementary strand of the target sequence, and then the DNA doubles.
The helix opens, at which point deaminase converts a single base: for CBE systems, cytosine deaminase changes cytosine to uracil; for ABE systems, adenine deaminase changes adenine to resemble guanine The I base will pair with the C base
.
After the base change, the bases of the two DNA strands are not paired, and the complementary strands are paired with new bases after DNA repair, thus realizing the replacement of a single base pair
.

Figure 14.
CBE technology

Figure 15.
ABE technology

Base editing technology has the characteristics of high editing efficiency and less editing damage, but the use of deaminase may increase the risk of carcinogenesis, and the possible off-target effects will seriously affect the editing efficiency
.

The off-target phenomenon of the BE system.
In 2019, the David R.
Liu team reported the development of a new gene editing technology, PE (prime editor), which not only solved the off-target problem, but also realized all 12 possible base conversions
.
In 2021, Caixia Gao's team cooperated with David R.
Liu's team to successfully establish and optimize a PPE (plant prime editing) editing system suitable for plants, realizing precise base editing of plant genomes
.

PE is prime editing.
In short, it is a method of introducing reverse transcription based on BE technology.
It can not only realize single base conversion, but also realize base knockout, addition and other operations
.
The principle of PE technology is by coupling Cas9 H840A nickase and reverse transcriptase together, and then under the guidance of a pegRNA (prime editing guide RNA) containing a segment of RNA, targeted binding and cleavage of a DNA strand, reverse transcription The generated single-stranded DNA will compete with the original sequence at the nick.
Since DNA repair enzymes generally bind the DNA strand from the 5 end, the original strand tends to be removed, and it is hoped that the added fragment is preferentially integrated into the original sequence.
In this way, base substitutions, additions and subtractions can be realized, and DNA fragments can also be added or subtracted
.

PE technology is still immature, and its reliability needs to be further studied, and similar to base editing technology, the use of reverse transcriptase and deaminase still has potential safety hazards
.

Figure 16.
PE technology

04.
CRISPR/Cas9 technology

The CRISPR/Cas9 system is mainly composed of Cas9 protein and single-stranded guide RNA (sgRNA).
Among them, Cas9 protein plays the role of cutting DNA double strands, and sgRNA plays the role of guide.
Under the guidance of sgRNA, through the principle of base complementary pairing, Cas9 protein Different target sites can be cleaved to achieve DNA double-strand breaks
.

Figure 17.
Main principles of CRISPR/Cas9 technology

The main process of CRISPR/Cas9 technology is that crRNA binds to Cas9, and the conformation of Cas9 nuclease changes, generating an energy channel that makes DNA easier to bind
.
The Cas9/crRNA complex can recognize the PAM (5'-NGG) site and lead to DNA unwinding, allowing the crRNA to find the DNA complementary strand adjacent to the PAM site
.
When Cas9 binds to the DNA sequence complementary to the crRNA adjacent to the PAM site, the bridge helix inside the REC lobe and the target DNA form an RNA-DNA heteroduplex structure
.
Recognition of PAM sites includes activation of HNH and RuvC nuclear breaks that can cause double-strand breaks (DSBs) in target DNA, resulting in DNA degradation
.
If the crRNA is not complementary to the target DNA, Cas9 will be released, looking for a new PAM site
.
Linear target genome breaks in DNA can be repaired by non-homologous end joining (NHEJ) or homology-mediated repair (HDR), while non-homologous end joining (NHEJ) can cause insertion or deletion errors to achieve The purpose of targeted knockout of a gene
.

In the process of genome editing using the CRISPR/Cas9 system, tracrRNA and crRNA can be fused into one RNA (sgRNA) for expression, which can also play a role in targeted splicing
.
Therefore, the CRISPR/Cas9 tool we use now only has two parts: Cas9 nuclease and sgRNA
.
Using this tool, we can edit and transform any gene very conveniently and quickly, such as gene knockout, knock-in, site-directed mutation, and so on
.

Figure 18.
Gene insertion application of CRISPR/Cas9 technology

05.
Main applications of CRISPR/Cas9 technology

1.
Knock-out

Under normal circumstances, when cells have DNA double-strand breaks, high-efficiency non-homologous end joining (NHEJ) is used to repair the broken DNA
.
However, during the repair process, mismatches of base insertion or deletion usually occur, resulting in frameshift mutations that render the target gene nonfunctional
.
The CRISPR/Cas9 system can use the Cas9 protein to cut the target genome to form DNA double-strand breaks, thereby achieving gene knockout
.

2.
Knock-in

When the DNA double-strand is broken, if a DNA repair template enters the cell, the broken part of the genome will undergo homologous recombination repair (HDR) according to the repair template, thereby realizing gene knock-
in.
The repair template consists of the target gene to be imported and the homology sequence (homology arm) upstream and downstream of the target sequence.
The length and position of the homology arm are determined by the size of the editing sequence
.
DNA repair templates can be linear/double-stranded deoxynucleotide chains or double-stranded DNA plasmids
.
HDR repair mode occurs at a low rate in cells, typically less than 10%
.

The advantages of CRISPR/Cas9 technology are a wide range of applications, higher gene editing efficiency, simple operation, low cost, limited by low homologous recombination efficiency, PAM sequence dependence and off-target effects
.

06.
Application of gene editing technology in gene therapy

From the first in vitro validation of ZFNs in 1996 to the emergence of CRISPR/Cas9 technology in 2012 and the subsequent vigorous development, gene editing technology has developed rapidly, the editing efficiency and accuracy have been continuously improved, and the application fields have been continuously expanded
.
It can not only be used for the study of expression regulation and gene function, the construction of cell animal models, the screening of oncogenes and drug targets, but also has a huge development prospect in gene therapy, providing new opportunities for single-gene genetic diseases, cancer and other diseases.
of treatment
.

Gene therapy achieves the purpose of treating diseases by introducing normal genes or editing and repairing defective genes
.
At present, the use of gene editing technology has been applied in the gene therapy of various diseases, such as single-gene genetic diseases, ophthalmic diseases, AIDS and tumors
.

Gene therapy achieves the purpose of treating diseases by introducing normal genes or editing and repairing defective genes
.

Figure 19.
Development history of gene editing technology

Sickle cell disease (SCD) is caused by a single-gene point mutation at codon 7 of the β-globin gene
.
Hoban et al.
used ZFNs to specifically target the β-globin gene and induce DNA cleavage in CD34+ hematopoietic stem and progenitor cells
.
When ZFNs were delivered into cells with an integrase-deficient lentiviral vector or an oligonucleotide donor, gene correction of the β-globin locus was effectively achieved
.
Hoban's research provides an important approach to gene therapy for sickle cell anemia
.

Gene editing technology is not only used in the treatment of genetic diseases, but also has major breakthroughs in non-hereditary diseases
.
Age-related macular degeneration (AMD) is the main cause of blindness in adults, and choroidal neovascularization (CNV) is its main pathological feature.
Angiogenic factors such as VEGFA (vascular endothelial growth factor) A, High expression of VEGFA) gene is the main cause of lesions
.
Kim et al.
introduced pre-engineered Cas9 ribonucleoprotein (RNP) into adult mouse eyes to inactivate the VEGFA gene in the retinal pigment epithelium; and found that cas9 RNPs effectively reduced choroidal neovascularization in a mouse model of AMD generated area
.
This study demonstrates the potential of CRISPR/Cas9 technology for localized treatment of non-hereditary degenerative eye diseases
.

Gene editing technology is not only used in the treatment of genetic diseases, but also has major breakthroughs in non-hereditary diseases
.

Gene editing technology has also made preliminary progress in the fields of AIDS and tumor treatment
.
The first application of targeted nucleases in humans is to use ZFNs technology to edit the CCR5 gene to resist HIV
.
The researchers extracted T cells from HIV patients and used ZFNs technology to interfere with the CCR5 gene in T cells.
Since the CCR5 gene is a coreceptor for most HIV strains, especially early infection strains, interfering with the expression of the CCR5 gene can resist HIV infection, indicating that the gene Editing technology may become a new direction for AIDS treatment
.

Gene editing technology has also made preliminary progress in the fields of AIDS and tumor treatment
.

Gene editing is used in tumor treatment , mainly in combination with immunotherapy, especially with CAR (chimeric antigen receptor) T cells.
This method has great development prospects in leukemia, lymphoma and some solid tumors
.
CARs include extracellular single-chain variable fragments of tumor cell-specific antigens and intracellular chimeric signaling domains, which can activate T cells and kill tumor cells
.
Ren et al.
used the CRISPR/Cas9 system to simultaneously destroy multiple gene loci, and the resulting TCR (T cell receptor) and HLA-I (HLA class I) deficient CAR-T cells can be used as universal CAR-T cells for immune Treatment; in addition to generating universal CAR-T cells, gene editing technology can also knock out genes encoding T cell inhibitory receptors or signaling molecules such as PD1 (programmed cell death protein 1) and CTLA4 (cytotoxic T lymphocyte-associated protein 4) , for the generation of enhanced CAR-T cells
.

tumor treatment

In 2016, Lu's team from West China Hospital of Sichuan University conducted a clinical experiment of CRISPR gene editing technology, isolated T cells from patients with metastatic non-small cell lung cancer, and used CRISPR/Cas9 technology to knock out the PD-1 gene in the cells.
It is expanded to a certain amount in vitro and then reinfused into the patient to achieve the purpose of killing tumor cells.
However, simply knocking out the inhibitory factor of T cells is a double-edged sword, and further research is needed to knock out these T cells for clinical use.
Whether the inhibitory factor can cause uncontrolled proliferation of cells or produce severe autoimmunity
.

Since BE technology is a precise base conversion without causing double-stranded DNA breaks, it is undoubtedly a very effective tool for gene therapy
.
β-thalassemia is caused by mutations in the globin gene (hemoglobin-beta, HBB), and the pathogenesis in China and Southeast Asia is mainly caused by mutations in HBB genes A to
G.
In 2017, the team of Sun Yat-sen University Huang Jun used single-base editing technology to edit the point mutation of HBB in immature human ternary embryos, that is, the base G was changed to A to correct the error
.
This study is the first to use BE technology to precisely repair the mutation site of a genetic disease, opening a new window for the treatment of neonatal β-thalassemia and even other genetic diseases
.
Chadwick's team also used BE technology to knock out the PCSK9 gene, thereby reducing plasma cholesterol levels
.

This study is the first to use BE technology to precisely repair the mutation sites of genetic diseases.

07.
Conclusion and Outlook

Rapid advances in gene editing technology have greatly enhanced our ability to make precise changes to the genomes of eukaryotic cells
.
Editable nucleases, especially CRISPR/Cas systems with higher editing efficiency, simplicity and low cost, have revolutionized our study of genome function
.
The emergence and continuous improvement of single-base editing technology has achieved precise conversion of a single base and reduced off-target efficiency
.

Although the current gene editing technology still faces the problems of off-target efficiency and potential immune response and other side effects, it is believed that with the cross-integration of multiple disciplines and the joint cooperation of scientists in the future, the new generation of gene editing technology will be simpler, more efficient and more accurate.
problems will be gradually solved
.
With the breakthrough of new and effective targets for diseases, especially those diseases that are difficult to be cured by traditional therapies, the purpose of curing diseases can be achieved by correcting disease-causing genes or introducing beneficial mutations
.
The clinical translation and applied research of gene editing technology is worth watching, which will be the key to the next generation of translational therapies and treatment paradigms, and will also promote the rapid development of personalized medicine
.

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