What is Gene Editing?
Gene editing is an emerging genetic engineering technology that can precisely modify specific target genes in an organism’s genome. Simply put, gene editing is the use of a modified protein as a tool to carry out targeted modification of a specified gene. The gene editing tools are sequence-specific endonucleases commonly composed of a sequence-specific DNA binding domain and a non-specific DNA modification domain. Once the DNA binding domain identifies the DNA target site on chromosomes, the DNA modification domain will cut the DNA at the target site, generating the DNA breaks, which induces DNA damage repair, and finally achieves targeted editing of the specified genome.
- The first generation of gene editing technology—ZFNs
ZFNs (zinc finger nucleases) are the first generation of genome editing technology, and their function is developed based on zinc finger proteins (ZFPs) with unique DNA sequence recognition. ZFNs consist of ZFPs with specific recognition sequences and the FokI endonuclease. Among them, the DNA recognition domain composed of ZFPs can recognize and bind to specific sites of DNA, while the cleavage domain composed of FokI can perform the cleavage function, and the combination of the two can make the double-stranded DNA break (DSB) of the target site. Cells can repair the broken DNA through homologous recombination (HR) and non-homologous end joining (NHEJ) repair mechanisms. HR may carry out restoration modification or insertion modification to the target site, while NHEJ is prone to cause insertion mutation or deletion mutation. Both can cause frameshift mutations, thus achieving the purpose of gene knockout.
- The second generation of gene editing technology—TALENs
TALENs (transcription activator-like effector nucleases) are another flexible and efficient targeted editing technology after ZFNs. TALENs are similar in structure to ZFNs, and are composed of two parts, specific DNA-binding protein—TALE (transcription activator-like effector), and FokI nuclease. TALE protein is secreted by the plant pathogen Xanthomonas, which can specifically recognize and bind to the sequence of host target genes in plant cells, thereby regulating the gene expression of the host. During gene editing, two TALE proteins recognize and bind DNA target sites, and the FokI nuclease is responsible for cutting the target DNA, resulting in DNA DSBs and inducing DNA damage repair mechanism of the cells, thereby realizing the editing of genome target sites.
The synthesis and assembly of TALENs are simpler and more flexible than that of ZFNs. The key is to synthesize the DNA sequence encoding the tandem repeat region of the TALE protein. In theory, the DNA sequences encoded by multiple TALE repeat units can be connected multiple times. But the synthesis of such highly repetitive sequences presents certain difficulties and challenges. At present, a variety of fast and easy methods to synthesize and assemble TALENs have been developed, such as Golden Gate (GG) assembly and rapid high-throughput solid-phase synthesis.
- The third generation of gene editing technology—CRISPR-Cas
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) is developed based on an immune system of prokaryotes (bacteria and archaea). Due to the advantages of simple synthesis, short cycle, flexible operation, and high efficiency, this technology has attracted much attention.
CRISPR-Cas works as follows:
1) sgRNA binds to Cas protein to form a ribonucleoprotein (RNP) complex.
2) The RNP complex locates the target site on the genome under the guidance of sgRNA.
3) Cas protein recognizes the protospacer adjacent motif (PAM), and cuts the DNA double-strand at the target site, resulting in a DSB.
4) DSB induces an emergency repair mechanism in cells: NHEJ or HDR.
5) In the vast majority of cases (>80%), cells adopt the NHEJ repair pathway, which randomly generates deletions or insertions of individual bases at the target site, resulting in a gene knockout model.
6) In rare cases (<20%), when homologous fragments exist in the cell, the cell adopts the HDR repair pathway, so that the target site is accurately repaired.
7) By introducing exogenous gene fragments or mutant bases into homologous fragments, a gene site-directed insertion model or a gene site-directed mutation model can be obtained.
- Comparison of three gene editing technologies
As a revolutionary gene editing technology, CRISPR/Cas has obvious advantages. Compared with ZFNs and TALENs, CRISPR/Cas is much less difficult to design and construct, with lower cost, shorter development cycle, and higher efficiency of targeted modification. In addition, CRISPR/Cas also has the advantages of multi-target editing and the ability of RNA editing, which is not available in the other two technologies. However, CRISPR/Cas has an obvious disadvantage. If there is no PAM near the target gene, the editing of the target gene cannot be achieved. As the longest-developed gene editing technology, ZFN has accumulated a large amount of clinical trial data, and is more mature in application to drug research and development.
Highlights in Gene Editing Technology Applications
- Advantages in treating genetic diseases
There are many types of drugs for the treatment of genetic diseases. The most important one is the drug for the treatment of genetic diseases based on adeno-associated virus (AAV) vectors. However, these drugs have two disadvantages: one is the high price, and the other is the limited packaging capacity of AAV vectors. Take Spark’s AAV gene therapy as an example, it delivers the correct RPE65 gene to retinal cells to treat Leber congenital amaurosis type 2, and is the first FDA-approved gene therapy. But it costs as much as $850,000/year. However, this method is ineffective for Leber congenital amaurosis 10 (LCA10), because the coding sequence of the CEP290 gene is 7.5kb long, which is far beyond the packaging capacity limit of AAV virus (4.7kb). While gene editing drugs can edit longer gene sequences at a much lower cost.
- Improve the production efficiency and function of CAR-T cells
Chimeric antigen receptor (CAR)-T cell therapy achieves the purpose of treating cancer by collecting T cells in the patient’s body, and then transforming them with viruses carrying the CAR gene and infusing them back into the human body. The original preparation method is time-consuming, difficult, costly, and may cause an autoimmune reaction in humans. Gene editing can significantly improve the production capacity and efficiency of CAR-T cells. In 2016, Haoyi Wang’ team used CRISPR/Cas9 technology to carry out double gene knockout of TRAC, B2M, and triple gene knockout of TRAC, B2M, and PD-1 on CAR-T cells, which greatly reduced the autoimmunity and improved antitumor activity of CAR-T cells significantly. In 2017, another research team also used CRISPR/Cas9 technology to knock out TCR and HLA-I-related genes, and no graft-versus-host disease (GVHD) was observed in the knocked-out CAR-T cases; in addition, there were also successful clinical reports of UCAR-T therapy.
- Bring light to HIV patients
In 2019, a team of researchers from Temple University and the University of Nebraska Medical Center said they were able to use CRISPR/Cas technology to destroy human immunodeficiency virus (HIV) in humanized mice, the first time humans have demonstrated that HIV can be cured.
That same year, Chinese scientists genetically edited human stem cells in vitro, making them naturally resistant to HIV, and transplanted them into an HIV-infected patient with a hematological tumor. The gene-edited cells continued to survive in the patients for more than a year without significant side effects, but the cells were few and couldn’t reduce the viral load of HIV in the blood.
- Advantages for use in diagnostic reagents
In the field of in vitro diagnostics (IVD), CRISPR technology has many advantages over PCR technology: CRISPR has excellent diagnostic specificity and can detect a single base mutation, which is very suitable for early screening of tumors, detection of tumor susceptibility genes and disease-causing genes. At the same time, CRISPR diagnostic technology is simple in operation and less dependent on equipment with a lower price, and it is suitable for field detection and detection in underdeveloped areas. In addition, CRISPR diagnostics have low requirements for operators with visualized results, so home-use CRISPR diagnostic kits are also the development direction of many companies.
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Building new model animals
Although primate disease models are very important for medical research and development, it was almost impossible to construct primate disease models by gene editing methods in the past. After the birth of CRISPR/Cas technology, people can easily operate gene editing on primates, thereby constructing various disease models.
The Development Trends of Gene Editing
- ZFNs and TALENs continue to reduce costs and shorten development cycles
Compared with CRISPR, the obvious disadvantages of ZFNs and TALENs are complex construction, long development cycle, and high cost. It is the main trend of the future development of these two technologies to make up for these defects in the future. The development cycle of ZFNs could be as long as three months, while identifying the final therapeutic ZFNs usually took a year. After improvements, the development cycle has been reduced to 10 days, during which the constructs are designed, assembled and tested in cells, and the total time required to finally produce therapeutic ZFNs has been reduced to 3 months.
- CRISPR gene editing is not restricted by PAM, enabling arbitrary genome editing
CRISPR-Cas9 has an obvious shortcoming that it cannot edit genes that are not near the PAM sequence. To overcome this hurdle, a research team led by biochemist Benjamin P. Kleinstiver from the MGH Center for Genomic Medicine designed a variant of the Cas9 protein that does not require a specific PAM to bind and cut DNA. The two new Cas9 variants, named SpG and SpRY, respectively, allow editing of DNA sequences with efficiencies not achievable with conventional CRISPR-Cas9.
- Overcome the off-target rate
Both ZFNs and CRISPR/Cas suffer from high off-target rates. In 2016, In 2016, a biotechnology company developed a system to select ZFNs that cleave genes at the correct location at a high rate. Even at a high level of on-target activity, the system guaranteed off-target rates down to detection limit level or lower. The key to the off-target rate of CRISPR/Cas lies in the design of gRNA. There are too many non-specific results between gRNA and non-target regions, and the off-target efficiency is high. In particular, the 8-10bp near the PAM cannot have high homology with the non-target region. At present, in order to improve the accuracy of genome editing, the Cas9 nuclease has been mutated. The mutated Cas can only cut one strand of the DNA double-strand. Two gRNAs guiding Cas to cut DNA can significantly improve the specificity of genome editing and reduce the off-target rate.