The paper recommended for you in this issue comes from an article in "pharmaceutical frontier" column of Journal of China Pharmaceutical University, Vol. 51, 2019, the research status and application progress of CRISPR / cas9 delivery system.
In view of the delivery forms of CRISPR / cas9 system at DNA, RNA and protein levels, this paper focuses on the research status of virus and non virus vectors of CRISPR / cas9 system, the new strategies of CRISPR / cas9 system delivery, and the application progress in biomedical field and gene related disease treatment. Through the summary and elaboration of CRISPR / cas9 system delivery and gene therapy strategy, it provides new ideas for the discovery of innovative drugs and the development of gene therapy.
Keywords: CRISPR / cas9 system; gene editing; viral vector; non viral vector; gene therapy
Article introduction
CRISPR / cas9 system is mainly composed of cas9 protein and single strand guided RNA (sgRNA). Cas9 protein has the function of DNA double strand cleavage, and sgRNA plays the role of guidance. In the presence of PMA adjacent to the prototype spacer, cas9 protein can reach different target sites through complementary base pairing under the guidance of sgRNA, and realize DNA double strand cleavage (DSB) by cutting target genes. There are two mechanisms of DSB repair: (1) non homologous end joining (NHEJ), NHEJ mediated repair can produce imprecise insertion and / or deletion mutations at DNA double strand break sites; (2) homologous recombination repair (HDR), Dr mediated precise repair can introduce precise point mutation or insertion mutation from single or double strand DNA donor templates (Figure 1).
At present, the delivery of CRISPR / cas9 system can be divided into three levels: plasmid, RNA and protein. Among them, the plasmids that deliver cas9 protein and sgRNA do not need to be transfected many times, which is convenient, fast and stable; the mRNA that delivers cas9 protein has lower miss rate compared with the delivery plasmids; the direct delivery of cas9 protein has certain advantages compared with the delivery methods of DNA or RNA, and its immunogenicity is low and there is no potential problem of permanently integrating CRISPR gene into the host genome.
Due to the high efficiency, multi-function and simplicity of CRISPR / cas9 system, it has become a new strategy in the biomedical field for the treatment of many diseases, such as the treatment of infectious diseases, the treatment of tumors, the establishment of human disease models and so on. However, due to the limitations of gene level modification, the development of CRISPR / cas9 system still needs further research to achieve its greater application value.
1. Delivery form of CRISPR / cas9 system
CRISPR / cas9 system is to realize the delivery of cas9 protein and sgRNA. The delivery form of cas9 protein can be divided into the following three types (Fig. 2): (1) deliver the plasmid expressing cas9 protein; (2) deliver the mRNA encoding cas9 protein; (3) directly deliver cas9 protein. Three delivery forms of CRISPR / cas9 system can reach the target site under the guidance of sgRNA to effectively achieve genome editing.
1.1 delivery of CRISPR / cas9 system from DNA level
Because of the advantages of high stability, simple operation and low cost, it is a common delivery form to deliver plasmids encoding cas9 protein and sgRNA from DNA level. However, the delivery of plasmids will reduce the efficiency of gene editing, prolong the cutting time of cas9 protein, and increase the miss rate. In addition, there are risks of gene integration.
1.2 delivery of CRISPR / cas9 system from RNA level
RNA level delivery is the delivery of mRNA and sgRNA encoding cas9 protein. MRNA only needs to be delivered to the cytoplasm and translated into cas9 protein under the function of ribosome in the cytoplasm. The limitation of this delivery method lies in the poor stability of mRNA itself and its easy degradation by RNase in vivo and in vitro. Therefore, a suitable delivery method is needed to protect mRNA from degradation by enzymes.
1.3 delivery of CRISPR / cas9 system from protein level
Delivery of cas9 protein and sgRNA is the most direct and simple method. This form has no transcription and translation process. Gene editing is faster and more efficient, with low miss rate and low toxicity. Cas9 protein and sgRNA can form riboprotein complex (RNP). Cas9 protein usually carries nuclear location signal (NLS) to help RNP enter the nucleus to complete gene editing. The difficulty of RNP delivery is that the RNP size is large and it is not easy to package, so it is very important to explore its suitable delivery carrier. Currently, the common carriers of RNP are liposomes, gold nanoparticles, cationic polymers and so on. In recent years, new carriers have been developed The development of ribosomes can also help RNP to escape and merge into nucleus, such as DNA nanoflower, zinc imidazole complex framework, cell microbubbles mediated by arrdc1, etc.
2. Virus vector of CRISPR / cas9 system
In recent years, CRISPR / cas9 virus vector has been widely used in vitro and in vivo because of its high transfection efficiency and stable transgene expression. Commonly used virus vectors include adeno-associated virus (AAV), adenovirus (ADV), lentivirus (LV) and phage.
2.1 adeno associated virus (AAV)
AAVS has been approved for some human clinical trials of gene enhancement therapy due to its good safety and therapeutic potential. Compared with other virus vectors, AAVS have lower immunogenicity, so AAVS is the most widely used virus vector for delivering CRISPR / cas9 system. The limitation of AAVS is that its encapsulation capacity is only 47 KB, and the large particle size of cas9 protein (spcas9) from Streptococcus pyogenes requires AAVS to deliver cas9 protein and sgRNA respectively. In view of the restriction of encapsulation size, smaller variants of cas9 protein have been studied. Ran et al. Screened out cas9 protein (sacas9) from Staphylococcus aureus, which has similar gene editing ability with spcas9, and has shortened more than 1KB.
2.2 adenovirus (ADV)
Adv is a double stranded DNA virus with icosahedral nucleocapsid structure, which has good biological characteristics, genetic stability, high gene transfer efficiency and large-scale usability.
2.3 lentivirus (LV)
The encapsulation capacity of lentivirus is about 8KB, which is a spherical structure composed of single stranded RNA. It is widely used in delivery CRISPR / cas9 system. LV is the same as adenovirus, which removes all genes and thus does not activate the immune system. LV as a delivery vector for CRISPR / cas9, host genome integration may lead to unnecessary non target insertion mutation, which has security risk. At present, most laboratories do not have the ability to prepare non genome integrated LV vectors, so Lv is less frequently used than AAV and adv, which is most commonly used to establish disease models.
2.4 phage
Phage vector is mainly used for anti multidrug resistant bacteria. Due to its narrow host selectivity, its research and application are relatively few.
3. Non viral vector of CRISPR / cas9 system
Although viral vectors can effectively deliver the non viral vector system of CRISPR / cas9 system through cell transduction, it may cause unnecessary immunogenicity and mutation risk in the host body, thus limiting its clinical transformation. In recent years, the research on the delivery of CRISPR / cas9 system by non viral vectors, especially nano carriers, has made some progress.
3.1 liposomes
At present, there are commercial liposome carriers on the market, such as lipofectaminetm, turbofectaminetm, ste, and fecttm, which have completed the delivery of CRISPR / cas9 system. On this basis, the development of new liposome carriers has also made preliminary progress.
3.2 polymer
Polymers are widely used in the delivery of genetic drugs. The polymer with positive charge can encapsulate the gene drugs by electrostatic attraction and nucleic acid with negative charge.
3.3 gold nanoparticles
Gold nanoparticles, as inorganic nanomaterials, have excellent chemical stability, good biocompatibility and large specific surface area.
3.4 other new non viral vectors
Sun et al. Prepared DNA nanoflower (dnanc) by the method of ring rolling replication. The purified cas9 protein and sgRNA formed RNP in vitro. RNP was combined with DNA nanoflower by the principle of complementary base pairing between sgRNA and DNA nanoflower. After successful encapsulation, Pei with positive charge was coated outside the DNA nanoflower to promote the escape of the inner body. In this study, u2osegfp was used as the report cell line, which confirmed that DNA nanoflower borne CRISPR / cas9 system could target and destroy EGFP gene under the guidance of sgRNA (Figure 3C).
Alsaiari et al. Made use of zifs, which contain imidazolyl and zinc ions, effectively achieved the encapsulation of large-size cas9 protein and sgRNA into cczif nanoparticles. The imidazolyl group in the carrier promoted the escape of cczif in vivo and transport to the nucleus under pH condition, and finally reduced the expression level of egpf protein by 37% (Fig. 3D).
In addition to the new nano carriers mentioned above, there are also inhibitors of protein domain protein 1 (arrdc1) - mediated microbubbles (stmms), graphene oxide (go) nano carriers and so on, which also realize the delivery of CRISPR / cas9 system.
4. CRISPR / cas9 system delivery new strategy
4.1 modification of cas9 protein and sgRNA
In order to achieve better delivery of CRISPR / cas9 system, the modification of cas9 protein and sgRNA has been widely studied. The modified cas9 protein and sgRNA can reduce the miss rate and increase the stability. In addition to the modification of cas9 protein, sgRNA can also be shortened from 20 nucleotides to 17 or 18 to reduce the miss rate.
4.2 cell penetrating peptide
Cell penetrating peptide (CPP), a kind of short peptide with the ability of transmembrane transport, has been used as a tool for efficient delivery of cas9 protein and sgRNA.
5. Application of the system in the field of Medicine
5.1 establishment of tumor disease model
CRISPR / cas9 system is mainly used for the establishment of disease models at cell level and animal level. In tumor research, the genetic defects of somatic cells are closely related to the etiology and pathological phenotype. In order to fundamentally seek the treatment of tumor, a variety of cell-level tumor models based on CRISPR / cas9 system have been established for pre clinical verification of tumor genes, drug targets and tumor drug resistance. At present, there are many tumor cell models based on CRISPR / cas9 system, including lung cancer cell line, breast cancer cell line and acute myeloid leukemia cell line.
5.2 antiviral research
The specific destruction of virus genes is a new way of antiviral strategy. CRISPR / cas9 system has become one of the first choice for various antiviral applications due to its relative versatility, specificity, flexibility and ease of use. At present, based on CRISPR / cas9 system, there have been studies on human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpes virus, human papillomavirus (HPV) and other viruses.
5.3 treatment of monogenic and polygenic diseases
Recent clinical trials have shown that gene therapy has been successful in the treatment of hereditary diseases. Hemophilia is a single gene linked recessive genetic disease. The mutation of fix gene will lead to the decrease of protein function and the abnormality of blood coagulation.
6. Summary and Outlook
Viral vector has high transfection efficiency, but it has high mutation rate and cancer risk, which makes the clinical application of viral vector face great challenges. In addition, due to the limited virus loading capacity, it is difficult to achieve the efficient delivery of CRISPR / cas9 system, so the development of new non viral delivery vectors is very important for the treatment of CRISPR / cas9 system. Non viral vectors are faced with drug loading difficulties, poor stability, high miss rate, targeted delivery, high immunogenicity and production difficulties