The Application of CRISPR/Cas9 System in Cervical Precancerous Lesions

17 Background : The clustered regularly interspaced short palindromic repeat 18 (CRISPR)/Cas9 system is becoming a promising gene therapy method. 19 Herein, we evaluated the therapeutic effect of CRISPR/Cas9 system in cervical 20 carcinogenesis, especially cervical precancerous lesions. transgenic


Methods:
In cervical cancer/pre-cancer cell lines, we transfected the 22 CRISPR/Cas9, transcription activator-like effector nuclease (TALEN), and zinc 23 finger nuclease (ZFN) plasmids, respectively. We used the cell apoptosis, cell 24 viability, and colony formation assays to examine the efficiency and specificity 25 of inhibition of cell apoptosis and growth between the different gene editing tools. 26 Western blotting was used to estimate the related protein expression. We used 27 xenograft formation assays to examine the ability of inhibition of cell growth in

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The gene editing tools mainly include zinc finger nuclease (ZFN) [8], 58 transcription activator-like effector nuclease (TALEN). and clustered regularly 59 interspaced short palindromic repeat (CRISPR/Cas9). All these gene editing 60 tools could induce targeted DNA double-strand breaks (DSBs) and edit targeted 61 genes by stimulating the DNA repair mechanisms [9]. With the improvements 62 of these gene-editing tools, gene therapy is becoming more precise and 63 effective. In previous studies, our team has demonstrated that these gene 64 editing techniques designed for HPV oncoprotein genes could effectively 4 influence targeted cells [10,11]. However, the comparison of the efficacy of 66 gene therapies in HPV infection disease is not identified yet. 67 For the CRISPR/Cas9 system, researchers only need to design the gRNA 68 complementary to the target DNA sequence, without any other component [8]. 69 The CRISPR/Cas9 system might be an ideal alternative to ZFN and TALEN for 70 inducing targeted gene editing because it is rapid and easy to design. It could 71 cause DSB at the specific site which could be fixed by the cells' self-repairing 72 system in the form of NHEJ (non-homologous end-joining)  provided from our previous study [20]. The sequence of gRNA-HPV16 E7-1 117 was 5'-GCTGGACAAGCAGAACCGGA-3', and the sequence of gRNA-HPV16 118 E7-2 was 5'-GAGACAACTGATCTCTACTG-3'. The ZFN (MA13 and MA14) 119 and TALEN (T512) plasmids were from our own laboratory, which was used in 120 primary experiments [10,11]. We cloned the sgRNAs into the pSpCas9(BB)-  The digested products were viewed in 2% agarose gels, and the gene editing 129 rate was calculated as follows: (1-(1-cleaved bands)1/2)*100.

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The results of the T7E1 assay and γ-H2AX confirmed that the CRISPR/Cas9  SiHa and S12 cells (Figure 2, k-l). In the S12 cell line, CRISPR/Cas9 was 264 observed to more effectively decrease E7 expression than the other two gene 265 editing tools. In addition, we found that the HPV16 E7 related protein 266 expressions have also be influenced. The expression of RB protein was 267 increased with the decrease of HPV16 E7 expression.

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To observe the dynamic changes during the therapy, we applied the vaginal 300 transfection of gRNA-E7-1+Cas9 in mice every 3 days, and sacrificed mice at 301 the different time points. After 12 days, we found that the expression of HPV16 302 E7 protein was decreased in cervical epithelia in IHC results. This trend became 303 increasingly obvious over time, and HPV16 E7 expression was almost invisible 304 on the 24th day. At the same time, we also observed the decreased expression 305 of P16 protein in cervical epithelial cells. The H&E results showed that in gRNA-306 E7-1+Cas9 treated mice the CIN of mice was gradually returned to normal-like 307 cervical epithelial with the increase of treatment time (Figure 4c). 308 We assigned the female K14-HPV16 positive mice randomly into 2 groups.

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For K14-HPV16 mice, previous studies have observed that the premalignant 348 stages of squamous carcinogenesis in the K14-HPV16 mice is highly similar to 349 that of humans [26], making it an ideal model for cervical precancerous lesions.

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CRISPR/Cas9 has been considered to have potential advantages for many 363 chronic pathogenic diseases caused by DNA viruses, which cannot be cured 364 using available drugs [27][28][29]. Compared with ZFN and TALEN, we observed 365 that CRISPR/Cas9 also has significant growth inhibitory and apoptotic effects 366 on cervical cancer and cervical pre-cancer cell lines and inhibited the tumor 367 formation of the S12 cell line in nude mice. In fact, the target sites of these 3 368 gene editing tools were not exactly the same point; thus, the comparison 369 between them also has its limits. However, the incomparably fast design 370 process, high scalability, and affordability make CRISPR/Cas9 an ideal gene 371 editing tool compared with ZFN and TALEN.

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The off-target effect and safety of CRISPR/Cas9 has always been the focus 373 of our concern. Through in vivo and in vitro experiments, we observed that

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A recent study showed that during the infectious period, the HPV16 genome 384 shared many amino acid-changing variants, while E7 protein was genetically 385 conserved [17]. In addition, HPV16 E7 is considered to be a single oncoprotein 386 that could cause cervical cancer in the animal model and immortalize human 387 keratinocytes in vitro [30,31]. This made HPV16 E7 an ideal target for the 388 therapy of HPV16-induced cervical cancer, which means that our 389 CRISPR/Cas9 system targeted for HPV16 E7 might have promising clinical 390 applications. In addition, the HPV16 E6 gene is also a good candidate cleavage 391 site for gene therapy, which has obtained promising results in vitro and in nude 392 mouse models [8,12]. We have also considered expanding the CRISPR/Cas9 393 cleavage sites of HPV16 in a follow-up study.