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Highlights • Next generation sequencing is a scalable solution to genotyping mutant mice. • Ratios of wild type and mutant sequence counts are used to call the genotype. • Hundreds of samples can be multiplexed into one sequencing experiment. • Amplification of high-homology genes can be easily filtered out during analysis., Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.

Graphical abstract, Highlights • Emergence of carbapenem-resistant S. algae is a severe problem. • Re-sensitization of S. algae to carbapenem by CRISPR/Cas9 genome editing. • The blaOXA-55-like gene is essential for carbapenem resistance in S. algae. • One-plasmid genome editing system for CRISPR/Cas9 genome editing in S. algae. • CRISPR/Cas9 genome editing is a promising approach to validate the gene function., Antibiotic resistance in pathogens is a growing threat to human health. Of particular concern is resistance to carbapenem, which is an antimicrobial agent listed as critically important by the World Health Organization. With the global spread of carbapenem-resistant organisms, there is an urgent need for new treatment options. Shewanella algae is an emerging pathogen found in marine environments throughout the world that has increasing resistance to carbapenem. The organism is also a possible antibiotic resistance reservoir in humans and in its natural habitat. The development of CRISPR/Cas9-based methods has enabled precise genetic manipulation. A number of attempts have been made to knock out resistance genes in various organisms. The study used a single plasmid containing CRISPR/Cas9 and recE/recT recombinase to reverse an antibiotic-resistant phenotype in S. algae and showed blaOXA-55-like gene is essential for the carbapenem resistance. This result demonstrates a potential validation strategy for functional genome annotation in S. algae.

Objective α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER “knock-in” mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. Methods We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3′ UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Results Tamoxifen injection of Gcg-CreERT2;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal. Conclusions We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types., Highlights • Developed a new Gcg-CreERT2 knock-in mouse line using CRISPR-Cas9 gene targeting. • Gcg-CreERT2 mice exhibit high recombination in α-cells and L-cells. • There is no evidence of preproglucagon haploinsufficiency in Gcg-CreERT2 mice.

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5′-6FAM 3′-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method., Highlights • A contamination-free visual detection method of SARS-CoV-2 with CRISPR/Cas12a is developed. • The detection process can be completed within 40 min with high sensitivity of as low as 20 copies RNA. • The detection results can be observed by our portable 3D printing instrument and smart phone. • For real respiratory swab samples detection, our detection results have 100% positive agreement and 100% negative agreement.

Highlights • We generated a floxed allele by using paired Cas9n (nickase), gRNAs and single DNA template in mouse. • We confirmed that the floxed allele was germline transmitted and functional in F1 offspring. • A floxed allele of the isoprenoid synthase containing domain (Ispd) gene in C57BL/6N background mice was created. • This method can be used to generate knockout mice for genes that are potentially embryonic lethal., CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.