Genome editing and its applications in genetic improvement in aquaculture

Purpose Colorectal cancer (CRC) is one of the major contributors to cancer mortality and morbidity. Finding strategies to fight against CRC is urgently required. Mutations in driver genes of APC or β-catenin play an important role in the occurrence and progression of CRC. In the present study, we jointly apply CRISPR/Cas9-sgRNA system and Single-stranded oligodeoxynucleotide (ssODN) as templates to correct a heterozygous ΔTCT deletion mutation of β-catenin present in a colon cancer cell line HCT-116. This method provides a potential strategy in gene therapy for cancer. Methods A Cas9/β-catenin-sgRNA-eGFP co-expression vector was constructed and co-transfected with ssODN into HCT-116 cells. Mutation-corrected single-cell clones were sorted by FACS and judged by TA cloning and DNA sequencing. Effects of CRISPR/Cas9-mediated correction were tested by real-time quantitative PCR, Western blotting, CCK8, EDU dyeing and cell-plated clones. Moreover, the growth of cell clones derived tumors was analyzed at nude mice xenografts. Results CRISPR/Cas9-mediated β-catenin mutation correction resulted in the presence of TCT sequence and the re-expression of phosphorylation β-catenin at Ser45, which restored the normal function of phosphorylation β-catenin including reduction of the transportation of nuclear β-catenin and the expression of downstream c-myc, survivin. Significantly reduced cell growth was observed in β-catenin mutation-corrected cells. Mice xenografted with mutation-corrected HCT-116 cells showed significantly smaller tumor size than uncorrected xenografts. Conclusion The data of this study documented that correction of the driven mutation by the combination of CRISPR/Cas9 and ssODN could greatly remedy the biological behavior of the cancer cell line, suggesting a potential application of this strategy in gene therapy of cancer.

/pdf/cas9-mediated-correction-of-b-catenin-mutation-and-restoring-23p6jy12ov.pdf

Cas9 Mediated Correction of β- catenin Mutation and Restoring the Expression of Protein Phosphorylation in Colon Cancer HCT-116 Cells Decrease Cell Proliferation in vitro and Hamper Tumor Growth in Mice in vivo

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3′-end and the WWP at the 5′-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques. 

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.

Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.

Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger's sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.

DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize.

Mpox is an infectious viral disease, and, as of January 12, 2023, about 84 560 distinct cases have been detected, involving about 110 countries since May 2022. Most clustered regularly interspaced short palindromic repeat (CRISPR)-based detection methods require long assay time because of the pre-amplification to target nucleic acids. Herein, we designed a new prototypal mpox biosensor that allows amplification-free nucleic acid detection by combining CRISPR/Cas12b system and ultrasensitive graphene field-effect transistor (gFET). The CRISPR/Cas12b–gFET enabled the detection of the mpox virus (MPXV) DNA targets at a sensitivity of ∼1 aM within about 20 min. We designed several sgRNAs to recognize the target genes of MPXV and then Cas12b protein cleaved the target genes to form a double-strand break. The specificity of sgRNAs of MPXV designed was verified among other homologous Orthopoxviruses, and the sgRNA could distinguish between recent 2022 MPXV and West African MPXV with the mutation site, showing its potential application in MPXV detection. Thus, the demonstrated CRISPR–gFET detection technology could serve as a sensitive and rapid diagnostic tool for MPXV and other DNA viruses in the future. 

Rapid and ultrasensitive detection of mpox virus using CRISPR/Cas12b-empowered graphene field-effect transistors

The invention relates to a method for rapidly preparing sgRNA by using a plurality of overlapping primers PCR method, which belongs to the field of molecular biology. The invention designs a pluralityof overlapping primers, amplifies and synthesizes a complete DNA fragment by PCR, and the complete DNA fragment is transcribed into sgRNA in vitro, and is combined with Cas9 enzyme to perform nucleicacid cleavage function. The invention uses multiple overlapping primers to synthesize DNA template of sgRNA in one step for the first time, and the experiment proves that the DNA template has high cleavage efficiency. The method established by the invention has value in the aspects of fast synthesis of sgRNA and reduction of experiment consumption.

Method for rapidly preparing sgRNA by PCR with multiple overlap primers

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a huge impact on the world, causing millions of deaths and a serious blow to the global economy. SARS-CoV-2 can attack most body tissues with devastating consequences [1]. The current global SARSCoV-2 outbreak has exposed our shortcomings in the prevention and control of infectious diseases, and there is an urgent need for rapid, large-scale and highly efficient diagnostic tools to detect infections in a timely and rapid manner with nucleic acids of virus (Fig. 1).

/pdf/mopcs-next-generation-nucleic-acid-molecular-biosensor-1nsmwwc0.pdf

MOPCS: next-generation nucleic acid molecular biosensor

Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

/pdf/customized-one-step-preparation-of-sgrna-transcription-cw0pwn8odx.pdf

Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing.

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Rapid and ultrasensitive detection of mpox virus using CRISPR/Cas12b-empowered graphene field-effect transistors

Method for rapidly preparing sgRNA by PCR with multiple overlap primers

MOPCS: next-generation nucleic acid molecular biosensor

Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing.