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A review of Nature Biotechnology, knocking at the door of CRISPR — the evolutionary history of gene editing

In recent years, CRISPR is considered to be the simplest and most efficient gene editing method, and it has also become one of the most rapidly developing emerging technologies in the history of biotechnology. In June 2022, on the 10th anniversary of CRISPR’s publication, Nature Biotechnology published a Reviw titled”knock in on crispr’s door”, which combed the achievements of scientists’ continuous exploration and breakthroughs in CRISPR gene editing technology in the past 10 years [1].

Figure 1 Nature Biotechnology issued a document in June 2022

CRISPR based gene therapy is in full swing. Gene therapy refers to the introduction of foreign normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, so as to achieve the purpose of treatment. Gene therapy with its unique potential to cure genetic diseases for life with one dose makes all impossibility possible. As of today, through the analysis of clinicaltrials According to gov, 56 clinical trials based on CRISPR are under way worldwide, including 21 in China, accounting for more than 30%. At present, most gene therapies are in vitro therapies (ex vivo), that is, cells are edited in vitro by CRISPR and then infused into the body to function. Common diseases such as tumor immunotherapy car-t, genetic diseases such as thalassemia, sickle anemia, hemoglobin genetic diseases and other blood diseases. In vivo, on the other hand, directly delivers therapeutic genes to the patient’s site to treat diseases. At present, it has shown great potential in congenital amaurosis, hereditary thyroid amyloidosis, hereditary angioedema and other diseases.

Figure 2 Global CRISPR clinical trial distribution hot spot map

Source:clinicaltrials gov

The development process of gene editing early gene editing ZFN and talen gene editing technology has mainly developed three generations. The early two generations of gene editing are mainly ZFN and talen. These two gene editing technologies are relatively simple and can be understood as”gene scissors” – restriction enzymes that cut specific DNA sequences. However, ZFN technology has obvious shortcomings, such as easy to miss the target, and may produce a series of unpredictable gene mutations, causing cytotoxicity. The emergence of talen technology has optimized the Miss target problem of ZFN technology to a certain extent, and has the advantages of simple design, higher specificity and activity. Therefore, talen technology has become a powerful tool in gene function research and gene therapy research. The fly in the ointment is that talen needs to repeatedly construct the fusion protein for different targets every time, which will cause some tedious work. The third generation of gene editing – crisprcrispr/cas9 is the third generation of”genome targeted editing technology” after ZFN and talen. Crispr/cas9 system consists of two parts:cas9 protein and guide RNA (single guide RNA, sgRNA). Cas9 protein has helicase activity, which can spin the DNA chain, and has endonuclease activity, which can cut the DNA chain. Its principle is that the endonuclease cas9 protein recognizes specific genomic sites through guide RNA (gRNA) and cleaves double stranded DNA to form DSB, and then realizes gene targeted knockout or insertion through HDR and NHEJ.

Figure 3 Crispr/cas9 schematic diagram [2]

Compared with traditional ZFN and talen technologies, crispr/cas9 technology is simpler. It only needs to construct sgRNA for specific sites, and its efficiency is higher than the previous technologies. It plays an increasingly important role in disease treatment research. However, crispr/cas9 system still has some limitations, which are mainly reflected in the dependence of the system on PAM sequence in DNA and the potential off target effect during cleavage. Therefore, based on crispr/cas9, scientists have developed more efficient and broad-spectrum accurate gene editing tools – single base editing technology be (base editor) and accurate gene editing tool PE (prime editors). Single base editing technology be (base editor) single base editing technology is a technology based on the fusion of deaminase and crispr/cas9 system. In 2016, David Liu Laboratory of Harvard University first reported the development of CBE single base editing tool. By fusing spcas9 with cytosine deaminase (CYD, such as Apobec1), the single base conversion from cytosine (c) to thymine (T) can be realized within a certain mutation window (Fig. 4) [3]. At the end of October 2017, the laboratory further developed the Abe single base editing tool, realizing the accurate conversion from adenine (a) to guanine (g) (Figure 5), providing a new research tool for gene editing [4].

Figure 4 CBE schematic [3]

Figure 5 Abe diagram [4]

Compared with crispr/cas9 technology, be technology can neither introduce DNA double strand breaks nor need recombinant repair templates, which improves the safety and accuracy of editing as a whole, and its efficiency is much higher than that of HDR and NHEJ repair caused by DSB. It has great application potential for many genetic diseases caused by point mutations. In recent years, several laboratories have also published similar tools and carried out more in-depth transformation and Optimization on the basis of these tools. Based on the combination of different single stranded DNA deaminase domains and cas9 notch enzyme, bangyao bioscientist team developed a new generation of DNA base editing tools – Ultra-high activity hycbes and double base editor a& C-bemax and other new base editing tools have improved editing activity and broadened the range of targets to achieve broader and more accurate gene editing. Relevant research results have also been published in international famous journals such as nature cell biology and Nature Biotechnology [5].

Figure 6 Hycbe schematic diagram of ultra-high precision base editor

Figure 7 Schematic diagram of double base editor precise gene editing tool PE (prime editors)

On October 21, 2019, David Liu Laboratory of Harvard University developed a new precise gene editing tool PE (prime editors) [6], which is based on crispr/cas9 system and optimized in two aspects:1 Pegrna:pegrna (prime editing guide RNA) is a modified sgRNA, which adds an RNA sequence at the 3’end of the traditional sgRNA. This RNA sequence includes a primer binding site (PBS), which is used to complement the cleaved target DNA strand; It also includes a sequence of RT template for reverse transcription, which is homologous with the DNA sequence downstream of the incision, and there are corresponding editing mutations (such as point mutations or insertion deletion mutations) in the RT sequence.

Figure 8 Modification of pegrna [4]2 Fusion protein:ncas9 (h840a) was fused with m-mlv reverse transcriptase.

Figure 9 PE structure diagram [4]

Under the guidance of pegrna, the fusion protein will reach the target sequence on the genome and cut the target DNA strand containing PAM (non complementary strand of pegrna). After that, the PBS sequence complements and pairs with the cut target DNA strand, and reverse transcriptase enlightens reverse transcription from the port vacancy. Reverse transcripts (DNA) contain the editing mutations we expect. This reverse transcriptional DNA will invade and enter genomic DNA, integrate and repair the incision. As long as RT sequence allows, this principle can be used to complete base point mutation (arbitrary conversion or transversion) and fragment insertion and deletion.

Figure 10 PE schematic diagram [4]

Compared with other gene editing tools (using ZFN, talen, cripsr/cas9, etc. to generate DSB for HDR or NHEJ repair or single base editing through base editing system), the advantage of PE is that it can achieve more accurate editing and a wider range of trials without relying on DSB. But at the same time, compared with CBE and Abe, PE’s disadvantages are also reflected. The editing efficiency is not as good as the former, and the possibility of producing random indels will also be improved.

Figure 11 Efficiency comparison of PE with Abe and CBE [6]

Finally, in addition to the above-mentioned gene editing tools, scientists also found a series of other proteins of CAS family except cas9, such as cas12, cas13, casx, etc. These new findings are expected to enable gene therapy to solve a wider range of genetic diseases and promote basic biomedical research and clinical gene therapy research.