Journal: Advanced Science

Article Title: KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition

doi: 10.1002/advs.202206098

Figure Lengend Snippet: Kmt2d mutation by CRISPR/Cas9 promotes AML in mice. A) Schematic experimental design for mouse modeling using CRISPR/Cas9 system. Trp53 −/− ; Cas9 mouse HSPCs were transduced with mCherry‐linked sg Kmt2d ‐sg Nf1 ‐sg Cas9 or sgScramble‐sg Nf1 ‐sg Cas9 , and then transplanted into sub‐lethally irradiated syngeneic mice. B) Kaplan–Meier survival curves of mice transplanted with Trp53 −/− ; Cas9 HSPCs transduced with sgScramble‐sg Nf1 ‐sg Cas9 (blue; n = 5), sg Kmt2d_ #1‐sg Nf1 ‐sg Cas9 (red, n = 5), or sg Kmt2d_# 2‐sg Nf1 ‐sg Cas9 (orange; n = 5). ** p < 0.01 (log‐rank test). C) WBC, Hb, and PLT counts of sgScramble and sg Kmt2d mice 2 months post‐transplant. Graph represents the mean ± SD; ** p < 0.01, *** p < 0.001, ns, not significant (unpaired two‐tailed t ‐test). D) Representative flow cytometric profiles showing the expression of CD11b/Gr‐1, B220/CD3ε and c‐Kit in bone marrow cells of sacrificed TNKC ( Trp53 −/− ; sg Nf1 ; sg Kmt2d; sg Cas9 ) mice. E) Representative images of histological analyses of blood, spleen, liver, and bone marrow of sacrificed TNKC mice. F) T7 endonuclease I assay on Kmt2d in bone marrow cells of sacrificed TNKC mice. G) Mutation analyses of the Kmt2d regions targeted by CRISPR/Cas9 of TNKC bone marrow cells. Representative Sanger sequences of single TA clones.

Article Snippet: sgRNAs were designed by the ATUM CRISPR gRNA Design tool ( https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Mutagenesis, CRISPR, Transduction, Irradiation, Two Tailed Test, Expressing, T7EI Assay, Clone Assay

Journal: Advanced Science

Article Title: A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

doi: 10.1002/advs.202105231

Figure Lengend Snippet: Specificity and efficiency evaluation of the CRISPR molecular system with pUC19 mutagens and the pCDH GFP gene. A) and B) Detailed information of all DNA‐RNP possible combinations for pUC19 mutated amplicons and pCDH amplicons analyzed by agarose gel electrophoresis. C) and D) TAE agarose gel electrophoresis analysis of CRISPR‐cleavage products for the pUC19‐0.3k and pUC19‐0.8k groups, related to Figure . E) TAE agarose gel electrophoresis of CRISPR‐cleavage products for the pCDH‐2k and pCDH‐5k groups, related to Figure . F) In vitro binding assays for evaluating the efficiency of CRISPR/dCas9 using CRISPR‐magnetic beads (MBs). G, H) Capture efficiency of different target sites calculated using the qPCR Ct results from Figure , Supporting Information, (* p < 0.05, ** p < 0.01, two‐tailed t ‐test, n = 3; error bars represent standard deviations). I) TAE agarose gel electrophoresis analysis evaluate the capture efficiency of GFP DNA samples using dRNP‐functionalized beads and treated with different post‐release methods. A DNA molecular weight marker ranging from 100 bp to 5000 bp was applied in all agarose electrophoresis runs.

Article Snippet: All of the sgRNAs targeting recombinant pUC19 [New England Biolabs (NEB), Ipswich, MA, USA] vectors (Origin, Mut1, Mut2, and Mut3), the green fluorescent protein (GFP) sequence in the pCDH (NEB) vector (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3), and exons 7 and 47 of human DMD were designed using DNA 2.0 (now ATUM) CRISPR DNA design tool.

Techniques: CRISPR, Agarose Gel Electrophoresis, In Vitro, Binding Assay, Magnetic Beads, Two Tailed Test, Molecular Weight, Marker, Electrophoresis

Journal: Advanced Science

Article Title: A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

doi: 10.1002/advs.202105231

Figure Lengend Snippet: A,B) SPR angular spectra of the dRNP‐functionalized CRISPR‐SPR‐Chips detecting DNA fragments that contain different target sites. C,D) SPR signals generated by CRISPR‐SPR‐Chips with or without a graphdiyne layer immobilized with different loaded amounts of dCas9 protein. E,F) Recombinant pUC19 amplicons (0.8 kb) were detected by the corresponding dRNP‐functionalized CRISPR‐SPR‐Chi(ps (Origin, Mut1, Mut2, and Mut3). G,H) The pCDH amplicons of 5 kb were detected by the dRNP‐functionalized CRISPR‐SPR‐Chips with different target sites (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3).

Article Snippet: All of the sgRNAs targeting recombinant pUC19 [New England Biolabs (NEB), Ipswich, MA, USA] vectors (Origin, Mut1, Mut2, and Mut3), the green fluorescent protein (GFP) sequence in the pCDH (NEB) vector (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3), and exons 7 and 47 of human DMD were designed using DNA 2.0 (now ATUM) CRISPR DNA design tool.

Techniques: CRISPR, Generated, Recombinant

Journal: Advanced Science

Article Title: A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

doi: 10.1002/advs.202105231

Figure Lengend Snippet: Real‐time SPR response of the dRNP‐functionalized CRISPR‐SPR‐Chips used to monitor the kinetic process of DNA fragments binding to the corresponding dRNP‐functionalized CRISPR‐SPR‐Chips. A, B) Binding kinetics of recombinant pUC19 amplicons (0.3 kb and 0.8 kb). C, D) Binding kinetics of pCDH amplicons (2 kb and 5 kb). (E) Binding kinetics of the pUC19‐Mut3‐0.8k amplicons in concentrations ranging from 1.6 ng µL −1 to 1000 ng µL −1 . (F) Binding kinetics of the pCDH‐GFP‐Pos2‐5k amplicons in concentrations ranging from 1.6 ng µL −1 to 1000 ng µL −1 . G, H) Limit of detection (LOD) test of pUC19‐Mut3‐0.8k and pCDH‐GFP‐Pos2‐5k amplicons ( n = 3; error bars represent standard deviations).

Article Snippet: All of the sgRNAs targeting recombinant pUC19 [New England Biolabs (NEB), Ipswich, MA, USA] vectors (Origin, Mut1, Mut2, and Mut3), the green fluorescent protein (GFP) sequence in the pCDH (NEB) vector (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3), and exons 7 and 47 of human DMD were designed using DNA 2.0 (now ATUM) CRISPR DNA design tool.

Techniques: CRISPR, Binding Assay, Recombinant

Journal: Advanced Science

Article Title: A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

doi: 10.1002/advs.202105231

Figure Lengend Snippet: Kinetic constants for the interaction of the CRISPR‐SPR‐Chip and DNA analytes

Article Snippet: All of the sgRNAs targeting recombinant pUC19 [New England Biolabs (NEB), Ipswich, MA, USA] vectors (Origin, Mut1, Mut2, and Mut3), the green fluorescent protein (GFP) sequence in the pCDH (NEB) vector (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3), and exons 7 and 47 of human DMD were designed using DNA 2.0 (now ATUM) CRISPR DNA design tool.

Techniques: CRISPR

Journal: Advanced Science

Article Title: A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

doi: 10.1002/advs.202105231

Figure Lengend Snippet: Analysis of healthy and DMD clinical samples for DMD‐associated dystrophin exon 7 and exon 47 deletions using CRISPR‐SPR‐Chips. A) Schematic of the dystrophin gene (DMD) with target exons. B) Results of CRISPR‐SPR‐Chip analysis for the deletion (absence) of targeted exons (+) in healthy and DMD clinical samples. C) Change in angle obtained from the SPR measurement of the dRNP‐DMD7‐CRISPR‐SPR‐Chip with patient sample A1 using gradient concentrations. D) Negative signal threshold of CRISPR‐SPR‐Chips. E) Change in angle obtained from the SPR measurement of the dRNP‐DMD7‐CRISPR‐SPR‐Chip with all samples. The negative signal threshold is the result from panel D. F) Change in angle obtained from the SPR measurement of the dRNP‐DMD47‐CRISPR‐SPR‐Chip with all samples. The negative signal threshold is the result from panel D. All data were collected from three independent experiments (*** p < 0.001, two‐tailed t ‐test, n = 3; error bars represent standard deviations).

Article Snippet: All of the sgRNAs targeting recombinant pUC19 [New England Biolabs (NEB), Ipswich, MA, USA] vectors (Origin, Mut1, Mut2, and Mut3), the green fluorescent protein (GFP) sequence in the pCDH (NEB) vector (GFP‐Pos1, GFP‐Pos2, and GFP‐Pos3), and exons 7 and 47 of human DMD were designed using DNA 2.0 (now ATUM) CRISPR DNA design tool.

Techniques: CRISPR, Two Tailed Test

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