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Objective To establish human and mouse pancreatic cancer cell strains stably expressing Cas9 protein, green fluorescent protein, red fluorescent proteins, luciferase-tdTomato, and to validate the activity of luciferase and gene editing of Cas9 function for pancreatic cancer research using luciferase and CRISPR/Cas9 system. Methods In human pancreatic cancer cells (AsPC-1, CFPAC-1, HPAC, BxPC-3, HS 766T, MIA PaCa-2, PANC-1, and SW 1990), and mouse pancreatic cancer cell (Pan02), the cells were infected with Cas9-expressing plasmid pLv-EF1α-Cas9m1.1-Puro, and single-cell clones were selected for culture and expansion. After extracting the total protein, Western blot verified the expression level of Cas9; Infected with fluorescent protein expression plasmids pLv-EF1α-EGFP, pLv-EF1α-mCherry, pLv-EF1α-tdTomato, pLv-EF1α-Luc2-tdT, and selected single cell clones stably expressing fluorescent proteins were cultured and amplified under fluorescence microscope. Cas9 stable expression cell line was selected to be infected with pLv-EF1α-Luc2-tdT, and the monoclonal culture of stable expression of fluorescent proteins was selected for expansion under fluorescence microscope. One of the cell lines were selected to be infected with Lv-EF1a-mCherry, and the mCherry-positive cells were sorted out by flow cytometry, and then the guide RNA of mCherry gene was then infected by lentivirus to target the mCherry gene, and after cell expansion, mCherry knockdown was detected by fluorescence microscope observation and flow cytometry; 5 BALB/c Nude mice were subcutaneously inoculated with MIA PaCa-2-Luc2-tdT cells (1.0×107/cells each), and imaged in vivo after 36 days. Results 48 human pancreatic cancer cell strains with stable Cas9 expression were screened(including 23 cells expressing Cas9m1.1, 25 cells expressing Cas9m1.1-Luc2-tdT),33 pancreatic cancer cell strains with stable expression of fluorescent proteins were screened (8 cells expressing EGFP, 7 expressing mCherry, and 9 each expressing Luc2-tdT and tdTomato). Cells expressing mCherry and Cas9 were infected with mCherry gRNA and mCherry was knocked down. In vivo imaging showed that both bioluminescence and fluorescence luminescence were present in MIA PaCa-2 cells expressing Luc2-tdT. Conclusions 33 pancreatic cancer cell strains with stable expression of fluorescent proteins are successfully established, in which the Luc2-tdT-expressing cell strains have luciferase activity; 48 pancreatic cancer cell strains with stable expression of Cas9 are successfully established, and the Cas9 protein has gene editing activity, gene editing activity varies depending on the original cell strains.

Copyright of Basic & Clinical Medicine is the property of Editorial Office of Basic & Clinical Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To observe the efficiency of Cas9 RNP technology for the preparation of B2M and PD-1 double genes knock-out primary human T lymphocytes (hereinafter referred to as T cells). Methods Single-guide RNA (sgRNA) was designed and cloned into vector plasmids. The effective sgRNAs were screened out by Sanger sequencing in HEK293T cells. B2M/PD-1 single and double genes knock-out T cells were constructed respectively and successively. The editing efficiencies were verified by Sanger sequencing, TA cloning and flow cytometry. Results The vector plasmids containing sgRNAs were successfully constructed and the high-efficiency sgRNAs (B2M sgRNA1 and PD-1 sgRNA1) were obtained. The editing efficiencies of B2M/PD-1 single gene knocked-out T cells were both up to 90%. In double genes knock-out T cells, the editing efficiencies at gene level were up to 90%, while the down-regulation rates of B2M and PD-1 protein reached 86% and 89%, respectively. Conclusion Cas9 RNP technology could be used to prepare B2M and PD-1 efficiently double genes knock-out T cells.

Objective To establish a mouse model of hepatocyte-specific TM6SF2 knockout, and to investigate the role of TM6SF2 in the development of nonalcoholic fatty liver disease (NAFLD). Methods The CRISPR/Cas9 technique and the Cre/LoxP strategy were used to establish a stable mouse model of hepatocyte-specific TM6SF2 knockout. The mice with hepatocyte-specific TM6SF2 knockout and the control mice were given a normal diet or a high-fat diet (HFD) for 16 weeks, and related indices were measured, including general status (body weight and liver weight), glucose metabolic indices (fasting blood glucose and insulin), and lipid metabolism (plasma triglyceride, cholesterol, and liver triglyceride). The t-test was used for comparison of normally distributed continuous data between two groups. Results Under the condition of HFD, compared with the control mice, the mice with hepatocyte-specific TM6SF2 knockout had significantly higher liver weight (2.235±0.175 g vs 1.258±0.106 g, t=4.789, P < 0.01) and liver index (4.970%±0.298% vs 3.210%±0.094%, t=5.630, P < 0.01), and the loss of the TM6SF2 gene in hepatocytes aggravated the abnormal level of alanine aminotransferase induced by HFD (62.517±1.526 U/L vs 25.991±5.947 U/L, t=5.949, P < 0.01). Compared with the control mice under the condition of normal diet or HFD, the mice with TM6SF2 knockout had a significant increase in plasma insulin level (normal diet: 37.203±0.836 mIU/L vs 34.835±0.426 mIU/L, t=2.520, P=0.025; HFD: 41.093±1.226 mIU/L vs 35.817±0.500 mIU/L, t=3.985, P=0.007), while there were no significant differences in the other indices associated with glucose metabolism (all P > 0.05). Under the condition of HFD, there were no significant differences in the levels of plasma triglyceride and cholesterol between the mice with hepatocyte-specific TM6SF2 knockout and the control group (P > 0.05), while the mice with hepatocyte-specific TM6SF2 knockout had a significant increase in the level of liver triglyceride compared with the control mice (23.969±0.978 mg/g vs 18.229±1.633 mg/g, t=3.015, P=0.024). Conclusion Hepatocyte-specific knockout of TM6SF2 can aggravate liver lipid accumulation and liver injury in mice with NAFLD. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Edulis Fungi is the property of Acta Edulis Fungi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro . The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

A new genome editing system named NgAgo-gDNA was invented using 5，phosphorylated single-stranded guide DNA (gDNA) of 24 nucleotides and Natronobacterium gregoryi Argonaute (NgAgo). This system outperformed the RNA-guided genome editing system CRISPR-Cas9 on several features. The success of the NgAgogDNA project demonstrated the importance of bioinformatics in biological research and will stimulate the development of genome editing systems. The NgAgo-gDNA project was initiated from searching homologs of TtAgo and PfAgo，two other enzymes from the AGO protein family. The authors used the software PSI-BLAST against the NCBI NR database to retrieve homologous protein sequences. After further analysis and filtering， they found the NgAgo protein (GenBank ： AFZ73749.1)，which works at the temperature of 37 冗.The key step in the NgAgogDNA project is to narrow down a great number of AGO homologous protein sequences to several candidates using bioinformatics methods for experimental validation of their functions. These bioinformatics methods were not explained in the published paper but could belong to the empirical methodology. An alternative but advanced methodology is to use machine learning algorithms (e.g. support vector machine or random forest) to modify AGO proteins which work at a temperature close to 37 冗.The future studies can be conducted in several fields using bioinformatics methods. First，the structural information of the NgAgo protein can be used to reveal the mechanism of the DNA and protein interaction. The sequence with structure comparison between NgAgo and TtAgo & PfAgo or other AGO proteins will help understand their molecular functions. Second， using the sequence or structure similarities，more RNA-or DNA-binding proteins can be retrieved from the public databases to help design new genome editing systems. Third，since RNAi (RNA interference) uses AGO to cleave double stranded RNAs，the guide-target complexes of AGO proteins need be studied to reveal the common mechanisms and differences between genome editing and RNAi. Fourth，a great number ol AGO genes from lower to higher organisms can be used to study the evolution ol AGO and the coevolution between the viruses and the hosts. [ABSTRACT FROM AUTHOR]

Almost all archaea and many bacteria achieve adaptive immunity through a diverse set of CRISPR-Cas systems. From 2012, Cas9 has been harnessed by thousands of laboratories for genome editing applications in a variety of experimental model systems. Cas9 is driving innovative applications from basic biology to biotechnology and medicine. However, numerous challenges still remain, such as limited targeting range, toxicity, and potential for off-target mutagenesis. Cpf1 represents a class 2/type V CRISPR RNA-guided endonuclease that is distinct from the type II CRISPR Cas9 nuclease. Future exploration of strategies using Cpf1 is expected to bring solutions for some of the biggest challenges facing genome editing. Here, we review the development and applications of Cpf1 for a variety of research or applications, and highlight challenges as well as future directions.