Journal: Viruses

Article Title: Genomic Characterization and gE/gI-Deleted Strain Construction of Novel PRV Variants Isolated in Central China

doi: 10.3390/v15061237

Figure Lengend Snippet: Construction and identification of the mutant virus SX1911-ΔgE/gI. ( A ) Strategy for constructing SX1911-ΔgE/gI using the CRISPR/Cas9 and LoxP systems. Two sgRNAs were designed to guide Cas9 to delete the gE and gI genes, and GFP was used for both positive and negative screening of mutant virus production. ( B ) Identification of SX1911-ΔgE/gI via IFA and PCR targeting the gE gene. ( C ) Multistep growth curve of SX1911 and SX1911-ΔgE/gI in Vero cells. ( D ) Plaque sizes of SX1911 and SX1911-ΔgE/gI in Vero cells. Data are presented as the mean ± SD, and an asterisk indicates a significant difference between SX1911 and SX1911-ΔgE/gI. ***: p < 0.001.

Article Snippet: sgRNAs targeting the gE and gI genes were designed using an online CRISPR tool ( https://www.genscript.com/gRNA-design-tool.html , accessed on 15 May 2021).

Techniques: Mutagenesis, Virus, CRISPR

Journal: bioRxiv

Article Title: A primate model organism for cardiac arrhythmias identifies a magnesium transporter in pacemaker function

doi: 10.1101/2025.05.28.655959

Figure Lengend Snippet: (Top) Results of deep organ-wide phenotyping of SLC41A2 -/- mice (C57BL/6NJ- Slc41a2 em1(IMPC)J /Mmjax) generated and analyzed by the Knockout Mouse Phenotyping Program (KOMP2) at The Jackson Laboratory , and reproduced here. The SLC41A2 -/- knockout is a CRISPR/Cas9-generated 467 bp deletion encompassing SLC41A2 exon 5 and a flanking 322 bp intronic region that includes the splice donor and acceptor. Deep phenotyping of 348 SLC41A2 -/- mice by the assays indicated surveying >50 traits revealed a single abnormality: an adult cardiovascular conduction system defect manifesting as a widened QRS complex on ECG (lone black diamond on right). Subsequent phenotyping has also detected isolated skin and vocalization abnormalities . Note that no other ECG abnormalities (black diamonds: heart rate, HR; RR interval, RR; PR interval, PR; ST segment, ST; abnormal rhythms, Abnormal ECG detected) were detected; the absence of HR and RR interval abnormalities are notable here because they indicate normal SA node pacemaker function in the SLC41A2 knockout. (Bottom) Quantification of the QRS interval in ECGs of adult SLC41A2 wild-type (WT) and homozygous SLC41A2 -/- knockout (HOM) female and male mice. n=164 female WT, 169 male WT, 10 female HOM, 5 male HOM; p-values (two-tailed Fisher’s exact test). ms, milliseconds.

Article Snippet: CRISPR/Cas9 single guide RNAs (sgRNAs) targeting the N-terminal coding region in an early common exon (exon 2) of human SLC41A2 were bioinformatically designed using CRISPR design tools from Synthego , Integrated DNA Technologies (IDT) , and CRISPick (Broad Institute) .

Techniques: Generated, Knock-Out, CRISPR, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: A primate model organism for cardiac arrhythmias identifies a magnesium transporter in pacemaker function

doi: 10.1101/2025.05.28.655959

Figure Lengend Snippet: (a) Structures of human SLC41A2 gene (top) and spliced mRNA (bottom) and the site of the introduced SLC41A2 gene knockout (KO) in human iPSC-derived sinoatrial node cells (iSANCs). The SLC41A2 KO (SLC41A2 -/- ) is an 118 bp deletion in exon 2, causing an early truncation and frameshift of the coding sequence , which was created in human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9-mediated gene targeting prior to their differentiation into iSANCs, as described in panel c and . Numbers, exons 1-10. Dark blue shading, protein coding sequence in mRNA; light blue shading, non-coding sequences (5’-untranslated region, 5’UTR; 3’ untranslated region, 3’UTR). (b) Model of the membrane topology of human SLC41A2 with 11 transmembrane (TM) domains, modified from Sahni et al. , based on flow cytometry of epitope tagged SLC41A2 heterologously expressed in DT40 chicken cells. Sequence comparisons of lemur, human, and mouse SLC41A2 are shown in . (c) Experimental scheme and timeline for constructing human SLC41A2 -/- iSANCs and analyzing the effects of the gene knockout (see Methods for details). (d) Immunofluorescence photomicrographs of wild-type iSANCs co-stained for SLC41A2 (green) and phospholamban (PLN, red), a sarcoplasmic reticulum (SR) transmembrane protein, with DAPI counterstain (blue, nuclei). Insets, close up of boxed region showing a single iSANC. Note colocalization of SLC41A2 with SR-marker PLN. No SLC41A2 staining was detected in SLC41A2 -/- iSANCs , as expected for the early truncation and frameshift deletion. Scale bar, 10 µm. n=3 technical replicates. (e) Functional calcium imaging with Fluo-4 AM of cultured wild-type control and SLC41A2 -/- iSANCs. Representative traces (left, amplitude normalized to F peak /F 0 ) showing the rhythmic calcium transients (spikes) of wild-type iSANCs and the slower firing rate of SLC41A2 -/- iSANCs. Violin plots quantify the effects of SLC41A2 -/- knockout on calcium transient firing rate, amplitude, time to peak, tau decay time, and duration at 50%. Dark dotted line, median; light dotted lines, quartiles. n= number of cells analyzed (28 WT, 37 SLC41A2 -/- iSANCs); p-values (two-tailed with Mann-Whitney test): *, <0.05; **, <0.005; ***, <0.0005; ****, <0.00005; ns, not significant. Scale bar for tracings, 1 second, 1 normalized units. (f) Functional magnesium imaging with Mag-Fluo-4 AM of cultured wild-type control and SLC41A2 -/- iSANCs. Representative traces (top, amplitude normalized to F peak /F 0 ) showing rhythmic magnesium transients of wild-type iSANCs (top left), and three examples of altered waveforms of SLC41A2 -/- iSANCs (bottom left, ectopic, stunted, and irregular mini-transients; top right, ectopic, stunted transients appearing to fuse with normal transients to produce an irregular rhythm; bottom right, transients with delayed time to peak, tau decay time, and duration at 50%). Irregular rhythm plot quantifies % of iSANCs that manifest mini-transients. Violin plots as above quantify the effects of SLC41A 2 -/- knockout on magnesium transient firing rate, amplitude, time to peak, tau decay time, and duration at 50%. n=number of iSANCs analyzed (27 WT, 30 SLC41A2 -/- iSANCs) (statistics and significance as above, except p-value for irregular rhythm plot determined by two-tailed Fisher’s exact test). Scale bar for tracings, 1 second, 0.2 normalized units. (g) Model of SLC41A2 and magnesium dynamics in human iSANCs, the cardiac pacemaker cells (modified from Weisbrod D et al ). Current models for cyclical firing of SAN cells (“automaticity”) invokes two cyclically-active and coupled cellular “clocks”: a voltage membrane clock and an intracellular, sarcoplasmic reticulum-based calcium clock – . The cardiac cycle begins by hyperpolarization activation of plasma membrane channel HCN4 that initiates the membrane clock (which generates the I f funny current and causes Na + influx, alone or in combination with other Na + influx channels including NACLN, TRPM7, TRPM4, and T-type calcium channels (CaT: CACNA1G and CACNA1H that result in Ca 2+ influx) and calcium clock (the SR-transmembrane ryanodine receptor, RYR1, which pumps Ca 2+ from the SR into the cytoplasm and activates the plasma membrane Na + -Ca 2+ exchanger (NCX, encoded by SLC8 ) that in turn exchanges the cytoplasmic Ca 2+ for Na + into the cell). The resulting increase in membrane potential produced by the coupled clocks activates plasma membrane L-type calcium channels (LTCC), causing a rapid but transient Ca 2+ influx (calcium transient, gold sunburst) and the upstroke of the action potential (phase 0 depolarization), triggering each cycle of cardiac contraction. Much like the dynamic regulation of calcium, we observed cyclical magnesium transients (blue sunburst) in wild-type iSANCs and slower and broader calcium transients and altered magnesium transient dynamics and waveforms in SLC41A2 -/- iSANCs. We hypothesize a third clock, the “magnesium clock”, which interacts with and influences the membrane and calcium clocks, and involves the SLC41A2 magnesium transporter and dynamic regulation of the intracellular distribution of Mg 2+ between the sarcoplasmic reticulum and cytosol. Transient high levels of cytosolic Mg 2+ counteract the Ca 2+ and thereby slow the pacemaker firing rate, perhaps through the known ability of Mg 2+ to directly compete with Ca 2+ for binding to the key L-type calcium channel (LTCC) – .

Article Snippet: CRISPR/Cas9 single guide RNAs (sgRNAs) targeting the N-terminal coding region in an early common exon (exon 2) of human SLC41A2 were bioinformatically designed using CRISPR design tools from Synthego , Integrated DNA Technologies (IDT) , and CRISPick (Broad Institute) .

Techniques: Gene Knockout, Derivative Assay, Sequencing, CRISPR, Membrane, Modification, Flow Cytometry, Immunofluorescence, Staining, Marker, Functional Assay, Imaging, Cell Culture, Control, Knock-Out, Two Tailed Test, MANN-WHITNEY, Activation Assay, Clinical Proteomics, Produced, Binding Assay

Journal: bioRxiv

Article Title: A primate model organism for cardiac arrhythmias identifies a magnesium transporter in pacemaker function

doi: 10.1101/2025.05.28.655959

Figure Lengend Snippet: (a) Nucleotide sequences of the sgRNAs (“multi-guide”) designed and used to target human SLC41A2 for gene knockout in iPSCs , and the Sanger sequencing primer (SP) used to verify the deletion. (b) Map of the first coding exon (exon 2) of human SLC41A2 showing the hybridization positions of each sgRNA (G1, 2, 3) and the sequencing primer (SP). Light blue, portion of the 5’-untranslated region (5’UTR) in exon 2. Dark blue, coding sequence. Enlarged region below shows Benchling alignment (Multiple Alignment using Fast Fourier Transform, MAFFT v. 7.505) of amplicons generated by Sanger sequencing of genomic DNA harvested from control WT iSANCs (similarly derived from parental wild-type iPSCs) and the SLC41A2 -/- iSANC knockout clone created and used in this study. Note CRISPR/Cas9 targeting resulted in a 118 bp deletion in exon 2 of SLC41A2 -/- iSANCs, producing a coding sequence deletion and frameshift at codon 4 resulting in a translation termination codon (STOP) five codons later (residue 9). (c) Fluorescence micrographs of control wild-type (top) and SLC41A2 -/- knockout (bottom) iSANCs co-stained for SLC41A2 (green) and sarcoplasmic reticulum marker PLN (red), with DAPI counterstain (nuclei, blue). SLC41A2 staining is absent in knockout mutant cells. Scale bar, 10 µm. n=3 technical replicates.

Article Snippet: CRISPR/Cas9 single guide RNAs (sgRNAs) targeting the N-terminal coding region in an early common exon (exon 2) of human SLC41A2 were bioinformatically designed using CRISPR design tools from Synthego , Integrated DNA Technologies (IDT) , and CRISPick (Broad Institute) .

Techniques: Gene Knockout, Sequencing, Hybridization, Generated, Control, Derivative Assay, Knock-Out, CRISPR, Residue, Fluorescence, Staining, Marker, Mutagenesis