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Benchling Inc crispr guide rna design tool
Crispr Guide Rna Design Tool, supplied by Benchling Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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CRISPR:

Article Title: Exploring the functions of JAKMIP1 in neuronal IL-6/STAT3 signaling and its relevance to chromosome 15q-duplication syndrome
Article Snippet: Microscope slides were imaged either on the upright DM4B LED ( Leica Microsystems ) or inverted EVOS FLoidTM ( Invitrogen TM), DMi8 widefield ( Leica Microsystems ) and TCS SP8 confocal microscopes ( Leica Microsystems ). .. A CRISPR guide RNA (gRNA) sequence was designed to exon 5 of transcript ENST00000282924.9 of the JAKMIP1 gene using the CRISPR tool available in Benchling . .. The gRNA and its complementary sequence were purchased from Integrated DNA Technologies with sticky ends to facilitate type IIS restriction enzyme cloning (full details of the cloning process are described in File S3).

Article Title: Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency
Article Snippet: .. A gRNA sequences for CYFIP1 (5’-CGCGTCCTCCAGAGTCACCT-3’), JAKMIP1 (5’-GTCGAAGAAAGGCCGGAGCA-3’) and STAT3 (5’-GCTGCTGTAGCTGATTCCAT-3’) were designed for using the CRISPR tool available in the Benchling software (Benchling [Biology Software] (2024)) and purchased from Integrated DNA Technologies (along with their complementary sequence) with sticky ends to facilitate type IIS restriction enzyme cloning using BbsI. .. The donor tags for the ORANGE constructs were designed using the Benchling software, using the gRNAs and fluorescent protein sequences (mNeonGreen for CYFIP1, mScarlet for JAKMIP1 and mGold for STAT3) obtained from FPbase ( ) and SnapGene ® (from Dotmatics; available at snapgene.com ).

Article Title: Rsc1 DNA-binding bromodomain drives RSC activity at A-rich promoters
Article Snippet: Sequences encoding these tags were added to RSC1 and RSC2 loci by two successive rounds of marker-less CRISPR/Cas9 genome editing, using pWS158 (Addgene 90517) and pWS171 (Addgene 90518) as Cas9 expression vectors, as previously described ( ). .. For each RSC1 and RSC2 , a protospacer adjacent motifs (PAM) sequence within 30 nucleotides of the start codon was selected and a guide RNA (gRNA) was designed using the Benchling CRISPR tool ( http://www.benchling.com ). gRNAs were cloned into the pWS082 (Addgene 90517) using annealed oligonucleotides by Esp3I Golden Gate assembly. ..

Article Title: Disrupting the LINC complex for treating laminopathy
Article Snippet: .. Human Guide RNA Sequences Potential guide RNA sequences to disrupt human SYNE1 KASH domain or SUN1 SUN domain were determined using CRISPR tool in Benchling software (Benchling Inc. USA) and are shown in Table 3. .. TABLE 3 Potential guide RNA sequences to target final exons in human SYNE1 (Nesprin-1) or SUN1 genes SEQ ID Gene Name ENSEMBL gene ID Chromosome Position Strand Sequence PAM CRISPR enzyme NO: SYNE1 ENSG00000131018 6 515330 − TCGTGTATCTGAGCATGGGG TGGAAT saCas9 44 SYNE1 ENSG00000131018 6 515335 − GCCATTCGTGTATCTGAGCA TGGGGT saCas9 45 SYNE1 ENSG00000131018 6 515340 + TCCACCCCATGCTCAGATAC ACGAAT saCas9 46 SYNE1 ENSG00000131018 6 515320 − GAGCATGGGGTGGAATGACC GGG spCas9 47 SYNE1 ENSG00000131018 6 515321 − TGAGCATGGGGTGGAATGAC CGG spCas9 48 SYNE1 ENSG00000131018 6 515330 − TCGTGTATCTGAGCATGGGG TGG spCas9 49 SYNE1 ENSG00000131018 6 515333 − CATTCGTGTATCTGAGCATG GGG spCas9 50 SYNE1 ENSG00000131018 6 515334 − CCATTCGTGTATCTGAGCAT GGG spCas9 51 SYNE1 ENSG00000131018 6 515335 − GCCATTCGTGTATCTGAGCA TGG spCas9 52 SYNE1 ENSG00000131018 6 515345 + CCCATGCTCAGATACACGAA TGG spCas9 53 SYNE1 ENSG00000131018 6 515333 + CCCGGTCATTCCACCCCATG TTTG Cpf1 54 SUN1 ENSG00000164828 7 873276 + TTTTTCTAACTGGGGCCATC CTGAGT saCas9 55 SUN1 ENSG00000164828 7 873285 − CCGATACAGACAGGTATACT CAGGAT saCas9 56 SUN1 ENSG00000164828 7 873266 + AACTTCGGATTTTTTCTAAC TGG spCas9 57 SUN1 ENSG00000164828 7 873267 + ACTTCGGATTTTTTCTAACT GGG spCas9 58 SUN1 ENSG00000164828 7 873268 + CTTCGGATTTTTTCTAACTG GGG spCas9 59 SUN1 ENSG00000164828 7 873280 − ACAGACAGGTATACTCAGGA TGG spCas9 60 SUN1 EN5G00000164828 7 873296 + CTGAGTATACCTGTCTGTAT CGG spCas9 61 SUN1 ENSG00000164828 7 873281 + TTCTAACTGGGGCCATCCTG TTTT Cpf1 62 SUN1 ENSG00000164828 7 873282 + TCTAACTGGGGCCATCCTGA TTTT Cpf1 63 SUN1 ENSG00000164828 7 873283 + CTAACTGGGGCCATCCTGAG TTTT Cpf1 64 SUN1 ENSG00000164828 7 873284 + TAACTGGGGCCATCCTGAGT TTTC Cpf1 65 PAM, protospacer adjacent motif; saCas9, Staphylococcus aureus Cas9; spCas9, Streptococcus pyogenes Cas9; Cpf1, CRISPR from Prevotella and Francisella 1.

Article Title: MLL2 facilitates long-range gene regulation through LINE1 elements
Article Snippet: For the multilineage differentiation, cells were grown on 0.1% gelatin-coated plates, using KnockOut TM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 μM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days, as previously described by . .. For the generation of MLL1 -KO, Cxcl12 L1-KO, Cnrip1 L1-KO and Tcstv3 L1-KO cell lines, two sg-RNAs were designed flanking the region to-be deleted for each line, using Benchling’s CRISPR tool ( https://www.benchling.com/crispr ). .. For each sgRNA, two oligonucleotides were synthesized (Supplementary Data 3), annealed and cloned into a CRISPR-Cas9 expression vector (pX330-hCas9-long-chimeric-grna-g2p, provided by Leo Kurian’s lab). mESC were transfected with CRISPR-Cas9 constructs using Lipofectamine TM 3000 (Thermo Fisher Scientific, L3000001).

Article Title: 10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.
Article Snippet: .. Benchling provides a CRISPR tool seamlessly integrated within a broader, cloud-based molecular biology platform. ..

Article Title: CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis
Article Snippet: .. A mutation-specific gRNA was designed using the Benchling CRISPR tool and applied in a biosensor platform incorporating a ferrocene-labelled mutant DNA probe. ..

Sequencing:

Article Title: Exploring the functions of JAKMIP1 in neuronal IL-6/STAT3 signaling and its relevance to chromosome 15q-duplication syndrome
Article Snippet: Microscope slides were imaged either on the upright DM4B LED ( Leica Microsystems ) or inverted EVOS FLoidTM ( Invitrogen TM), DMi8 widefield ( Leica Microsystems ) and TCS SP8 confocal microscopes ( Leica Microsystems ). .. A CRISPR guide RNA (gRNA) sequence was designed to exon 5 of transcript ENST00000282924.9 of the JAKMIP1 gene using the CRISPR tool available in Benchling . .. The gRNA and its complementary sequence were purchased from Integrated DNA Technologies with sticky ends to facilitate type IIS restriction enzyme cloning (full details of the cloning process are described in File S3).

Article Title: Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency
Article Snippet: .. A gRNA sequences for CYFIP1 (5’-CGCGTCCTCCAGAGTCACCT-3’), JAKMIP1 (5’-GTCGAAGAAAGGCCGGAGCA-3’) and STAT3 (5’-GCTGCTGTAGCTGATTCCAT-3’) were designed for using the CRISPR tool available in the Benchling software (Benchling [Biology Software] (2024)) and purchased from Integrated DNA Technologies (along with their complementary sequence) with sticky ends to facilitate type IIS restriction enzyme cloning using BbsI. .. The donor tags for the ORANGE constructs were designed using the Benchling software, using the gRNAs and fluorescent protein sequences (mNeonGreen for CYFIP1, mScarlet for JAKMIP1 and mGold for STAT3) obtained from FPbase ( ) and SnapGene ® (from Dotmatics; available at snapgene.com ).

Article Title: Rsc1 DNA-binding bromodomain drives RSC activity at A-rich promoters
Article Snippet: Sequences encoding these tags were added to RSC1 and RSC2 loci by two successive rounds of marker-less CRISPR/Cas9 genome editing, using pWS158 (Addgene 90517) and pWS171 (Addgene 90518) as Cas9 expression vectors, as previously described ( ). .. For each RSC1 and RSC2 , a protospacer adjacent motifs (PAM) sequence within 30 nucleotides of the start codon was selected and a guide RNA (gRNA) was designed using the Benchling CRISPR tool ( http://www.benchling.com ). gRNAs were cloned into the pWS082 (Addgene 90517) using annealed oligonucleotides by Esp3I Golden Gate assembly. ..

Software:

Article Title: Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency
Article Snippet: .. A gRNA sequences for CYFIP1 (5’-CGCGTCCTCCAGAGTCACCT-3’), JAKMIP1 (5’-GTCGAAGAAAGGCCGGAGCA-3’) and STAT3 (5’-GCTGCTGTAGCTGATTCCAT-3’) were designed for using the CRISPR tool available in the Benchling software (Benchling [Biology Software] (2024)) and purchased from Integrated DNA Technologies (along with their complementary sequence) with sticky ends to facilitate type IIS restriction enzyme cloning using BbsI. .. The donor tags for the ORANGE constructs were designed using the Benchling software, using the gRNAs and fluorescent protein sequences (mNeonGreen for CYFIP1, mScarlet for JAKMIP1 and mGold for STAT3) obtained from FPbase ( ) and SnapGene ® (from Dotmatics; available at snapgene.com ).

Cloning:

Article Title: Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency
Article Snippet: .. A gRNA sequences for CYFIP1 (5’-CGCGTCCTCCAGAGTCACCT-3’), JAKMIP1 (5’-GTCGAAGAAAGGCCGGAGCA-3’) and STAT3 (5’-GCTGCTGTAGCTGATTCCAT-3’) were designed for using the CRISPR tool available in the Benchling software (Benchling [Biology Software] (2024)) and purchased from Integrated DNA Technologies (along with their complementary sequence) with sticky ends to facilitate type IIS restriction enzyme cloning using BbsI. .. The donor tags for the ORANGE constructs were designed using the Benchling software, using the gRNAs and fluorescent protein sequences (mNeonGreen for CYFIP1, mScarlet for JAKMIP1 and mGold for STAT3) obtained from FPbase ( ) and SnapGene ® (from Dotmatics; available at snapgene.com ).

Clone Assay:

Article Title: Rsc1 DNA-binding bromodomain drives RSC activity at A-rich promoters
Article Snippet: Sequences encoding these tags were added to RSC1 and RSC2 loci by two successive rounds of marker-less CRISPR/Cas9 genome editing, using pWS158 (Addgene 90517) and pWS171 (Addgene 90518) as Cas9 expression vectors, as previously described ( ). .. For each RSC1 and RSC2 , a protospacer adjacent motifs (PAM) sequence within 30 nucleotides of the start codon was selected and a guide RNA (gRNA) was designed using the Benchling CRISPR tool ( http://www.benchling.com ). gRNAs were cloned into the pWS082 (Addgene 90517) using annealed oligonucleotides by Esp3I Golden Gate assembly. ..

other:

Article Title: Solid phase transitions as a solution to the genome folding paradox.
Article Snippet: Ultra-long-range genomic contacts, which are key components of neuronal genome architecture, constitute a biochemical enigma.. This is because regulatory DNA elements make selective and stable contacts with DNA sequences located hundreds of kilobases away, instead of interacting with proximal sequences occupied by the exact same transcription factors.. This is exemplified in olfactory sensory neurons (OSNs), in which only a fraction of LHX2-, EBF1and LDB1-bound sites interact with each other, converging into highly selective multi-chromosomal enhancer hubs.

Mutagenesis:

Article Title: CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis
Article Snippet: .. A mutation-specific gRNA was designed using the Benchling CRISPR tool and applied in a biosensor platform incorporating a ferrocene-labelled mutant DNA probe. ..



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<t>CRISPR/Cas9</t> shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).
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<t>CRISPR/Cas9</t> shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).
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<t>CRISPR/Cas9</t> shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).
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Image Search Results


CRISPR/Cas9 shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 shows high editing efficiency in PGCs. (A) Phase-contrast images of cultured PGCs isolated from embryonic blood, showing typical colony morphology after 3 weeks in vitro . Left: male PGC colony; right: female PGC colony. Scale bar = 20 µm. (B) Immunofluorescence for germ cell markers in PGCs. These cells (male line shown) strongly express SSEA-1 (green, cell surface) and VASA/DDX4 (red, cytoplasm), even after long-term culture (>50 days). Nuclei are counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative fluorescence microscopy of EGFP + PGCs 5 days after co-electroporation with Cas9 and sgRNAs targeting EGFP (gEGFP1+2). Left: cells electroporated with Cas9 mRNA at 1 µg, 2 µg, or 3 µg (with constant gRNA amount). Right: cells electroporated with Cas9 protein (RNP complex) at equivalent molar doses (1:1.2 Cas9:sgRNA ratio). In both mRNA and protein conditions, higher Cas9 doses result in loss of EGFP fluorescence and reduced cell numbers (rounding and death) compared to lower doses. Scale bar = 20 µm. (D) Flow cytometry analysis of EGFP fluorescence and cell viability in edited versus control PGCs. Left: histogram overlays of EGFP intensity for control (untreated EGFP + PGCs, gray) vs. CRISPR-edited cells (green). Cas9-edited populations shift toward lower fluorescence, indicating EGFP knockout. Upper right: bar graph quantifying the percentage of EGFP + cells in each group (mean ± SEM, n = 3). Both Cas9 mRNA and Cas9 protein treatments caused a dose-dependent decrease in the fraction of EGFP-expressing cells compared to control (p-values are indicated in the figure by one-way ANOVA). Lower right: plot showing the percentage of live cells recovered during flow cytometry. Higher Cas9 doses correlate with reduced live-cell recovery, reflecting CRISPR-induced cytotoxicity in PGCs. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: CRISPR, Cell Culture, Isolation, In Vitro, Immunofluorescence, Fluorescence, Microscopy, Electroporation, Flow Cytometry, Control, Knock-Out, Expressing, Cell Recovery

CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

CRISPRi has limited efficacy in gene knockdown in PGCs. (A) Schematic of the CRISPR interference (CRISPRi) system. (i) The PGK-CRISPRi-EGFP plasmid expresses dCas9-KRAB (catalytically inactive Cas9 fused to the KRAB repressor) and an EGFP marker under a constitutive PGK promoter. (ii) The gCAG-mCherry plasmid carries a U6.3 promoter–driven sgRNA targeting the CAG promoter and a CAG-driven mCherry reporter. (iii) Co-transfection strategy: dCas9-KRAB (plasmid i) is expressed in the cell, and the sgRNA (plasmid ii) guides it to the CAG promoter in the mCherry cassette, silencing mCherry transcription. (B) Summary of CRISPRi reporter knockdown efficacy in human 293T cells vs. chicken cells. Bars show the percentage of mCherry + cells in each condition (no sgRNA, mock control, +gCAG sgRNA). In 293T cells, introducing the CAG-targeting sgRNA significantly reduces the mCherry + fraction relative to controls, whereas in DF-1 cells the mCherry + percentage remains unchanged, and in PGCs only a slight decrease is observed. (C) Expression of the dCas9-KRAB-EGFP fusion protein in CRISPRi. Western blot confirmed that dCas9-KRAB-EGFP is only expressed in CRISPRi cells, indicating the successful construction of CRISPRi stable PGC cell lines. Blank: Untransfected cells served as the negative control. (D) Gene expression following CRISPRi-mediated knockdown in CRISPRi cells. qRT-PCR showed no significant reduction in expression of the target genes for which CRISPRi sgRNAs were designed. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPRi has limited efficacy in gene knockdown in PGCs. (A) Schematic of the CRISPR interference (CRISPRi) system. (i) The PGK-CRISPRi-EGFP plasmid expresses dCas9-KRAB (catalytically inactive Cas9 fused to the KRAB repressor) and an EGFP marker under a constitutive PGK promoter. (ii) The gCAG-mCherry plasmid carries a U6.3 promoter–driven sgRNA targeting the CAG promoter and a CAG-driven mCherry reporter. (iii) Co-transfection strategy: dCas9-KRAB (plasmid i) is expressed in the cell, and the sgRNA (plasmid ii) guides it to the CAG promoter in the mCherry cassette, silencing mCherry transcription. (B) Summary of CRISPRi reporter knockdown efficacy in human 293T cells vs. chicken cells. Bars show the percentage of mCherry + cells in each condition (no sgRNA, mock control, +gCAG sgRNA). In 293T cells, introducing the CAG-targeting sgRNA significantly reduces the mCherry + fraction relative to controls, whereas in DF-1 cells the mCherry + percentage remains unchanged, and in PGCs only a slight decrease is observed. (C) Expression of the dCas9-KRAB-EGFP fusion protein in CRISPRi. Western blot confirmed that dCas9-KRAB-EGFP is only expressed in CRISPRi cells, indicating the successful construction of CRISPRi stable PGC cell lines. Blank: Untransfected cells served as the negative control. (D) Gene expression following CRISPRi-mediated knockdown in CRISPRi cells. qRT-PCR showed no significant reduction in expression of the target genes for which CRISPRi sgRNAs were designed. Statistical significance was determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: The amplicons were subjected to Sanger sequencing, and sequencing traces were analyzed using the Inference of CRISPR Edits (ICE) software tool (v3.0, Synthego).

Techniques: Knockdown, CRISPR, Plasmid Preparation, Marker, Cotransfection, Control, Expressing, Western Blot, Negative Control, Gene Expression, Quantitative RT-PCR