The progress and challenges of gene editing technology and the application of gene therapy.

Abstract: Gene editing is a technology that specifically alters the target sequence of genetic material.
in recent years, zinc finger nuclease (ZFN), transcriptionactivating factor effect nuclease (TALEN), irregular clusters of interval short-echo repetition (CRISPR) and single-base editing (BE) technology have emerged, not only for gene function research provides a powerful tool, but also provides a new treatment plan for life medicine.
gene editing technology has been widely used in animal cell model construction, drug target screening and gene function research, etc.
this paper summarizes the progress of gene editing technology and its application in gene therapy, and discusses the principles, development history, advantages and disadvantages of gene editing technology, as well as the prospects and opportunities of application in gene therapy, in order to provide reference for the clinical transformation of gene editing technology.
gene editing technique is a technology for the purpose of changing the gene sequence for the purpose of achieving fixed-point mutation, insertion or knockout.
has been exploring gene editing technology since the end of the 20th century, but it wasn't until 2013 that CRISPR/Cas9 technology was successfully used in mammalian cells that it greatly contributed to the development of gene editing technology.
the genomeof seukual organisms contain billions of bases, and the operation of their genomes has been challenging.
homologous recombination technology (homologous recombination, HR) is the earliest gene editing technology and a major breakthrough in eyrepheutic gene editing. the principle of
is to introduce the exogenous gene into the receptor cell, through the exchange of homologous sequence, so that the exogenous DNA fragments replace the gene at the site, so as to achieve the purpose of inactivating or repairing defective genes of a particular gene.
but for higher eyre organisms, the natural recombination rate of exogenous DNA and target DNA is very low, only 1/10E7 to 1/10E6;
to meet this challenge, a series of nuclease-based gene editing techniques have emerged to enable precise and effective gene editing in eyrepharycations, especially in mammals.
compared with traditional gene editing techniques, nuclease-based gene editing technology reduces random insertion of exexternal genes and increases the chance of precise modification of specific fragments of the genome.
the current gene editing technology mainly includes the following: artificially mediated zinc finger nuclease technology (zinc finger nucleases, ZFNs), transcription activator-effect nuclease technology (quail-type activator-effects of the factor stales suates, TALENs), regular clusters of intervals repeat the relevant protein technology (CRISPR/Cas9) and single-base editing technology. The research boom caused by gene editing technology in
is, on the one hand, because of the development of gene editing technology itself, and more accurate, efficient and low-cost gene editing technology is constantly being developed, on the other hand, gene editing technology, as an important tool, plays an important role in basic research such as gene screening, animal and cell model construction, and also provides new ideas for gene therapy for many diseases.
therefore, this paper summarizes the development of gene editing technology and its exploration and application in gene therapy, and discusses the challenges and opportunities of gene editing technology.
1, gene editing technology research progress based on the rapid development of DNA nuclease gene editing technology, from the first generation of DNA nuclease editing system ZFNs, the second generation TALENs to the third generation CRISPR/Cas9 system, gene editing efficiency is increasing, the cost is gradually reduced, the application range is expanding.
three gene editing techniques, such as ZFNs, TALENs, and CRISPR/Cas9, were established on the basis of the double-strand breaks of DNA (double-strand breaks, DSBs) at the genomic target site, which in turn activates the internal repair mechanism of the cell.
the repair mechanisms for DNA double-stranded fractures in cells include easy to cause random insertions, missing heterogenend end connections (non-homologous end joining, NHEJ) and homology recombination repair (homology directed repair, HDR) that requires the presence of a homono template to be activated.
2016, BE technology was developed to enable single base conversion without causing DNA double-stranded fractures and without homologous templates, effectively circumventing the deficiencies of gene editing techniques based on NHEJ and HDR repair after double-stranded DNA fractures.
1.1 ZFNs In the 1990s, the discovery of Fok I enzymes contributed to the emergence of ZFNs.
1996, the Chandrasegaran team at Johns Hopkins University's Department of Environmental Health Sciences in the United States developed ZFNs technology based on fokI enzyme and zinc finger protein fusion.
ZFNs contain two domains: the DNA-bound zinc finger protein region and the restricted nucleic acid endoenzyme FokI. nuclease incision active region (Figure 1).
zinc finger protein region determines the sequence specificity of ZFNs.
zinc finger base sequence is generally composed of 30 amino acids, the conservative region of which binds zinc ions is usually 4 cysteine or 2 cysteine and 2 histaline, and its spatial structure consists of 1 alpha helix and 2 reverse beta parallel structures.
1, 3, and 6-bit amino acids of alpha helix specifically identify and bind to three consecutive bases in the DNA sequence.
because the 1, 3, and 6 amino acids of alpha helix in different zinc finger base sequences are different, the zinc finger protein region consisting of 3 to 6 different zinc finger-based sequences is connected to the FokI nuclease region to form an artificial nuclease that can specifically identify DNA sequences and cut.
FokI. must be dipolymerd in advance to be active.
because FokI's own dipolymerization can also cut DNA, but the cutting efficiency is low and easy to produce non-specific cutting, so in the design of ZFNs, FokI can be mutated, so that it can not form homologous dipolymer.
when two mutations combining different target sequences, FokI. are separated by spacers of 5 to 7 bp, which can form an active heterogenous dipolymer with nuclease.
designed zFNs in this way can increase the specificity of their DNA sequence recognition.
based on Chandrasegaran's work, Dana Carrol' team in the Department of Biochemistry at the University of Utah School of Medicine used ZFNs to inject fruit fly embryos into the embryo, the first time that gene editing was achieved in animals.
, scientists used ZFNs to edit target genes in animal, plant and human cells.
Although ZFNs technology has been successfully genetically edited in multiple species, the design of zinc finger proteins is time-consuming and costly, limiting the large-scale application of the method.
1.2 TALENs In 2009, the Adam J. Bogdanove team in the Department of Plant Pathology and Bioinformatics at Iowa State University in the United States and ulla Bonas team at the Institute of Biology at Martin Luther University in Germany, respectively, discovered the interaction of transcription-activated proteins (drillion-carper-like-effector, TALE) and DNA from plant-causing jaundice genus.
combines the TALE protein with the FokI enzyme region to build a new generation of nuclease editing technology - TALENs.
the composition of TALENs and ZFNs are similar in that they also contain FokI nuclease domain at the end of their pyrethic acid, except that the DNA binding domain of TALENs is TALE protein (Figure 1). Each identification module in the
TALE protein consists of 34 amino acids, with the exception of the 12th and 13th amino acids, the rest of the amino acid sequence is conservative, the 12th and 13th amino acids are called variable double amino acid residues (repeat di-residue, RVD).
RVD determines the DNA base that TALE identifies and binds.
4 different bases have corresponding TALE identification modules.
therefore, when constructing TALEN artificial nuclease, it is only necessary to connect the sequence of the different TALE identification modules in the order of the target sequence and then fuse with the coding sequence of FokI.
compared to ZFNs, TALENs are designed to be easy, and any DNA sequence can theoretically be designed and constructed with a specific TALEN nuclease.
but each base of the target sequence requires a TALE identification module, so the build process of TALENs is heavy. in addition, TALENs have lower cytotoxicity in human cells
.
2011, Miller and others used TALENs for the first time in human cells to edit the NTF3 and CCR5 genes, demonstrating the regulation and modification of the endogenous target gene by TALEN nuclease.
1.3 CRISPR/Cas9 2012 CRISPR/Cas9 in vitro reconstruction and 2013 demonstrated its gene editing function in human cells, marking the beginning of a new generation of gene editing.
the CRISPR/Cas9 system is derived from the naturally acquired immune system of bacteria and archaic bacteria, and is resistant to invasion of exogenous DNA through a complex of CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA) and Cas9 proteins. The basic process of
the CRISPR/Cas9 system can be divided into three stages: the first stage is the interval sequence acquisition period, the PLAS or phage-carrying DNA fragment is cut into a short DNA fragment by the host's nuclease, the eligible DNA fragment is integrated into the host CRISPR site to become the interval sequence between the crRNA repeat sequences; Cas9 protein expression period, Cas9 protein expression, CRISPR sequence processed by pre-crRNA into mature crRNA, mature crRNA contains interval sequences that are targeted to foreign invasion DNA;
The CRISPR/Cas system can be divided into five categories depending on the Cas protein: type I, III, and IV CRISPR sites contain a complex formed by crRNA and multiple Cas proteins;
many CRISPR systems rely on PAM (proto-spacer adjacent motif) sequences near crRNA target sites, and the absence of PAM sequences will lead to self-cutting of Type I and II CRISPR systems.
the CRISPR/Cas9 system, widely used for gene editing, is type II CRISPR, consisting of Cas9 protein and sgRNA (single guide RNA).
sgRNA is designed from the advanced structure of crRNA and tracrRNA formation, binds to Cas9 nuclease proteins, guides its identification and editing of target sequences, and the presence of A PAM-based sequence containing NGG or NAG near the target sequence (Figure 1).
compared to ZFNs and TALENs, CRISPR/Cas9 improves the specificity of Cas9 nuclease by identifying target sites using a wizard RNA-guided nuclease to match the target DNA fragments, while Cas9 functions as a monomer protein under the guidance of sgRNA, unlike The FokI.enzyme of ZFNs and TALENs, which is only if the dipolymerization of ZFNs and TALENs is used to eliminate the need to cut the dna to the dna of the complex.
however, because CRISPR/Cas9 comes from the defense system of the naturalacquired immune system of pronuclear organisms against foreign genetic material, Cas9 nuclease may inherit the characteristics of low sequence specificity, increasing the chance of nonspecific cutting, resulting in increased off-target effects.
different scientific teams have proposed different ways to modify or edit Cas9 and sgRNA to reduce off-target effects.
such as combining Cas9 proteins with FokI nuclease, zinc finger proteins or TALE proteins to improve The specificity of Cas9, the use of inactivated Cas9 proteins and FokI. regions to form new nucleases, making them active only when nuclease dipolymerization, fatih, etc., fusion of zinc finger proteins or cells with Cas9 protein variants to enhance nucleonuclease specificity.
another way to reduce off-target effect is to use incision enzyme instead of nuclease, resulting in single-chain fracture instead of double-stranded fracture, single-chain fracture can not induce the repair of NHEJ, can still activate the precise repair of HR.
single-chain fracture can reduce off-target effect, but also reduce the repair efficiency, so some people proposed the use of double incision enzyme, not only to improve the specificity of gene editing and improve the efficiency of editing.
1.4 BEZFNs, TALENs, and CRISPR/Cas9 technologies all rely on NHEJ and HDR that induce double-stranded fractures at the target site and activate DNA.
NHEJ is prone to random insertion and deletion, resulting in transcoding mutations, which affect the function of the target gene, HDR is more accurate than NHEJ, but its homologous recombination repair efficiency in cells is low, about 0.1% to 5%. the emergence of
BE technology has effectively improved the above problems.
April 2016, Harvard University's David Liu Laboratory published for the first time a gene-editing technique that does not require DNA double-stranded fractures or homologous templates to perform single-base conversion.
the technology.