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Testing of two different gRNAs to induce double-strand breaks in the IGH locus, in the absence of a donor template (A) Apheresis RM WBCs were thawed, and enriched B cells were stimulated for 2 days in culture. At day 3, the cells were electroporated in the presence of a <t>CRISPR-Cas9</t> gRNA complex targeting the IGH locus. Genomic DNA was isolated 2 and 5 days post-editing, PCR-amplified, and sequenced. (B) The Sanger sequencing was analyzed by <t>ICE</t> to quantify the indels. Two gRNAs targeting the IGH locus were tested (gRNA 1 and gRNA 2) in B cells from healthy donor animals (uninfected) or from SHIV-infected and ART-treated animals (SHIV + ART-suppressed). Two-tailed paired t tests were performed. p values are shown in the figure. (C) The MMEJ and NHEJ indel signatures were quantified at 2 days and 5 days post-editing. The data shown are representative of three different experiments in three different donors. Paired t tests were performed. p values are shown in the figure. (A) was generated using BioRender. Statistical analyses were performed using Prism software.

Crispr Edits Ice Software By Synthego, supplied by Synthego 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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1) Product Images from "Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production"

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2025.101598

Figure Legend Snippet: Testing of two different gRNAs to induce double-strand breaks in the IGH locus, in the absence of a donor template (A) Apheresis RM WBCs were thawed, and enriched B cells were stimulated for 2 days in culture. At day 3, the cells were electroporated in the presence of a CRISPR-Cas9 gRNA complex targeting the IGH locus. Genomic DNA was isolated 2 and 5 days post-editing, PCR-amplified, and sequenced. (B) The Sanger sequencing was analyzed by ICE to quantify the indels. Two gRNAs targeting the IGH locus were tested (gRNA 1 and gRNA 2) in B cells from healthy donor animals (uninfected) or from SHIV-infected and ART-treated animals (SHIV + ART-suppressed). Two-tailed paired t tests were performed. p values are shown in the figure. (C) The MMEJ and NHEJ indel signatures were quantified at 2 days and 5 days post-editing. The data shown are representative of three different experiments in three different donors. Paired t tests were performed. p values are shown in the figure. (A) was generated using BioRender. Statistical analyses were performed using Prism software.

Techniques Used: CRISPR, Isolation, Amplification, Sequencing, Infection, Two Tailed Test, Generated, Software

CRISPR-Cas9 RNA-guided editing in the IGH locus leads to VRC01 knockin (A) Schematic of the engineered VRC01 antibody and its VH4a promoter. (B) B cells were isolated from healthy donor (uninfected) RM. The cells were stimulated for 2 days in culture before electroporation in the presence of a CRISPR-Cas9 gRNA complex followed by transduction 30 min later with an scAAV-6 virus encoding the donor template. The genomic DNA was isolated 5 and 8 days post-editing, PCR-amplified, and sequenced. The Sanger sequencing was analyzed by ICE to quantify the indels. This figure is representative of three independent experiments using B cells from three different donors. Paired t test was performed using Prism with a resulting p value of 0.3514. ns, nonsignificant. (C) Schematic of the in-out PCR. Arrows: forward and reverse primers; UHA: upstream homology arm; DHA: downstream homology arm; VH4a: B-cell-specific promoter; VRC01: engineered anti-HIV bNAb; SS: splicing site. (D) VRC01 in-out PCR demonstrates VRC01 cassette knockin at the expected location in the IGH locus. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus.

Figure Legend Snippet: CRISPR-Cas9 RNA-guided editing in the IGH locus leads to VRC01 knockin (A) Schematic of the engineered VRC01 antibody and its VH4a promoter. (B) B cells were isolated from healthy donor (uninfected) RM. The cells were stimulated for 2 days in culture before electroporation in the presence of a CRISPR-Cas9 gRNA complex followed by transduction 30 min later with an scAAV-6 virus encoding the donor template. The genomic DNA was isolated 5 and 8 days post-editing, PCR-amplified, and sequenced. The Sanger sequencing was analyzed by ICE to quantify the indels. This figure is representative of three independent experiments using B cells from three different donors. Paired t test was performed using Prism with a resulting p value of 0.3514. ns, nonsignificant. (C) Schematic of the in-out PCR. Arrows: forward and reverse primers; UHA: upstream homology arm; DHA: downstream homology arm; VH4a: B-cell-specific promoter; VRC01: engineered anti-HIV bNAb; SS: splicing site. (D) VRC01 in-out PCR demonstrates VRC01 cassette knockin at the expected location in the IGH locus. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus.

Techniques Used: CRISPR, Knock-In, Isolation, Electroporation, Transduction, Virus, Amplification, Sequencing

B cell editing leads to VRC01 bNAb expression (A) Total RNA was extracted from edited B cells 5 days post-electroporation and -transduction. VRC01 mRNA amplification was analyzed by reverse transcription PCR. (B) VRC01 bNAb at the cell surface was also detected by flow cytometry at 8 days post-editing using an antibody directed against the Strep-tag II linker (Strep-Tactin PE). This experiment is representative of three independent experiments using three different donor animals. A summary of the three replicates is shown in (C). (D and E) The secreted VRC01 absorbances (D) and concentrations (E) were measured by ELISA using a secondary antibody directed against the Gly-Ser-StrepII linker (D) or anti-monkey IgG secondary antibody (E). B cell supernatants from 5 days (d5) and 8 days (d8) post-editing were either undiluted or diluted 1:2 or 1:4. The data are representative of a single experiment. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 virus transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus. Paired t test statistics were performed using GraphPad Prism. ns, nonsignificant; ∗ p < 0.05, ∗∗ p < 0.01.

Figure Legend Snippet: B cell editing leads to VRC01 bNAb expression (A) Total RNA was extracted from edited B cells 5 days post-electroporation and -transduction. VRC01 mRNA amplification was analyzed by reverse transcription PCR. (B) VRC01 bNAb at the cell surface was also detected by flow cytometry at 8 days post-editing using an antibody directed against the Strep-tag II linker (Strep-Tactin PE). This experiment is representative of three independent experiments using three different donor animals. A summary of the three replicates is shown in (C). (D and E) The secreted VRC01 absorbances (D) and concentrations (E) were measured by ELISA using a secondary antibody directed against the Gly-Ser-StrepII linker (D) or anti-monkey IgG secondary antibody (E). B cell supernatants from 5 days (d5) and 8 days (d8) post-editing were either undiluted or diluted 1:2 or 1:4. The data are representative of a single experiment. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 virus transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus. Paired t test statistics were performed using GraphPad Prism. ns, nonsignificant; ∗ p < 0.05, ∗∗ p < 0.01.

Techniques Used: Expressing, Electroporation, Transduction, Amplification, Reverse Transcription, Flow Cytometry, Strep-tag, Enzyme-linked Immunosorbent Assay, Virus, CRISPR

CRISPR:

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production
Article Snippet: The remaining PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, Germantown, MD, #28104), and 25 ng of purified DNA mixed with 3 μL of 3 μM IgH primers was submitted for Sanger sequencing by the Genomics Fred Hutch Shared Resources core. .. The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA). ..

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Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: Testing of two different gRNAs to induce double-strand breaks in the IGH locus, in the absence of a donor template (A) Apheresis RM WBCs were thawed, and enriched B cells were stimulated for 2 days in culture. At day 3, the cells were electroporated in the presence of a CRISPR-Cas9 gRNA complex targeting the IGH locus. Genomic DNA was isolated 2 and 5 days post-editing, PCR-amplified, and sequenced. (B) The Sanger sequencing was analyzed by ICE to quantify the indels. Two gRNAs targeting the IGH locus were tested (gRNA 1 and gRNA 2) in B cells from healthy donor animals (uninfected) or from SHIV-infected and ART-treated animals (SHIV + ART-suppressed). Two-tailed paired t tests were performed. p values are shown in the figure. (C) The MMEJ and NHEJ indel signatures were quantified at 2 days and 5 days post-editing. The data shown are representative of three different experiments in three different donors. Paired t tests were performed. p values are shown in the figure. (A) was generated using BioRender. Statistical analyses were performed using Prism software.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: CRISPR, Isolation, Amplification, Sequencing, Infection, Two Tailed Test, Generated, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: CRISPR-Cas9 RNA-guided editing in the IGH locus leads to VRC01 knockin (A) Schematic of the engineered VRC01 antibody and its VH4a promoter. (B) B cells were isolated from healthy donor (uninfected) RM. The cells were stimulated for 2 days in culture before electroporation in the presence of a CRISPR-Cas9 gRNA complex followed by transduction 30 min later with an scAAV-6 virus encoding the donor template. The genomic DNA was isolated 5 and 8 days post-editing, PCR-amplified, and sequenced. The Sanger sequencing was analyzed by ICE to quantify the indels. This figure is representative of three independent experiments using B cells from three different donors. Paired t test was performed using Prism with a resulting p value of 0.3514. ns, nonsignificant. (C) Schematic of the in-out PCR. Arrows: forward and reverse primers; UHA: upstream homology arm; DHA: downstream homology arm; VH4a: B-cell-specific promoter; VRC01: engineered anti-HIV bNAb; SS: splicing site. (D) VRC01 in-out PCR demonstrates VRC01 cassette knockin at the expected location in the IGH locus. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: CRISPR, Knock-In, Isolation, Electroporation, Transduction, Virus, Amplification, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production

doi: 10.1016/j.omtm.2025.101598

Figure Lengend Snippet: B cell editing leads to VRC01 bNAb expression (A) Total RNA was extracted from edited B cells 5 days post-electroporation and -transduction. VRC01 mRNA amplification was analyzed by reverse transcription PCR. (B) VRC01 bNAb at the cell surface was also detected by flow cytometry at 8 days post-editing using an antibody directed against the Strep-tag II linker (Strep-Tactin PE). This experiment is representative of three independent experiments using three different donor animals. A summary of the three replicates is shown in (C). (D and E) The secreted VRC01 absorbances (D) and concentrations (E) were measured by ELISA using a secondary antibody directed against the Gly-Ser-StrepII linker (D) or anti-monkey IgG secondary antibody (E). B cell supernatants from 5 days (d5) and 8 days (d8) post-editing were either undiluted or diluted 1:2 or 1:4. The data are representative of a single experiment. Mock is mock electroporated; transduced is mock electroporated followed by AAV6-VRC01 virus transduction; transduced + edited is electroporated with the CRISPR-Cas9 and gRNA complex followed by transduction with AAV6_VRC01 virus. Paired t test statistics were performed using GraphPad Prism. ns, nonsignificant; ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The analyses were performed using Inference of CRISPR Edits (ICE) software by Synthego (Redwood City, CA).

Techniques: Expressing, Electroporation, Transduction, Amplification, Reverse Transcription, Flow Cytometry, Strep-tag, Enzyme-linked Immunosorbent Assay, Virus, CRISPR

Genome intronic CRISPR gRNA sites and UFlip targeting vector homology arm sequences.

Journal: eLife

Article Title: Cre/ lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

doi: 10.7554/eLife.71478

Figure Lengend Snippet: Genome intronic CRISPR gRNA sites and UFlip targeting vector homology arm sequences.

Article Snippet: Amplicons were Sanger sequenced and the sequences analyzed for indel efficiency using Synthego’s Inference of CRISPR Edits (ICE) analysis software ( https://ice.synthego.com/#/ ).

Techniques: CRISPR, Plasmid Preparation

( A ) Diagram of the UFlip. The vector contains a floxed rox loxP lox2272 gene trap plus secondary marker loxP lox2272 rox cassette. The cassette is flanked by cloning sites for homology arms (HA) complementary to a genomic CRISPR target site, and universal gRNA sites (UgRNA) for in vivo liberation of the targeting cassette. ( B ) Gene ‘off’ alleles are generated by integration of the UFlip cassette into an intron in the active orientation, leading to transcription termination and splicing of the primary transcript in the mRFP gene trap. ( C ) Gene ‘on” alleles are generated by integration of the UFlip cassette into an intron in the passive orientation. This is driven by cloning the genomic 5’ homology arm downstream of the UFlip cassette, and cloning the genomic 3’ homology arm upstream of the UFlip cassette. Integration at the genomic CRISPR-Cas9 target site occurs in the opposite orientation. During transcription RNA polymerase reads through the integrated UFlip cassette, which is spliced out with the intron during processing of the primary transcript. ( D ) Cre-mediated recombination at an ‘off’ allele locks the cassette in the ‘on’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. ( E ) Cre-mediated recombination at an ‘on’ allele locks the cassette in the ‘off’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. BFP, blue fluorescent protein; gcry1 , gamma crystallin 1 promoter; myl7 , cardiac myosin light chain 7 promoter; 2 A, porcine teschvirus-1 2A peptide; mRFP, monomeric red fluorescent protein; pA, transcription termination and polyadenylation signal; SA, splice acceptor.

Journal: eLife

Article Title: Cre/ lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

doi: 10.7554/eLife.71478

Figure Lengend Snippet: ( A ) Diagram of the UFlip. The vector contains a floxed rox loxP lox2272 gene trap plus secondary marker loxP lox2272 rox cassette. The cassette is flanked by cloning sites for homology arms (HA) complementary to a genomic CRISPR target site, and universal gRNA sites (UgRNA) for in vivo liberation of the targeting cassette. ( B ) Gene ‘off’ alleles are generated by integration of the UFlip cassette into an intron in the active orientation, leading to transcription termination and splicing of the primary transcript in the mRFP gene trap. ( C ) Gene ‘on” alleles are generated by integration of the UFlip cassette into an intron in the passive orientation. This is driven by cloning the genomic 5’ homology arm downstream of the UFlip cassette, and cloning the genomic 3’ homology arm upstream of the UFlip cassette. Integration at the genomic CRISPR-Cas9 target site occurs in the opposite orientation. During transcription RNA polymerase reads through the integrated UFlip cassette, which is spliced out with the intron during processing of the primary transcript. ( D ) Cre-mediated recombination at an ‘off’ allele locks the cassette in the ‘on’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. ( E ) Cre-mediated recombination at an ‘on’ allele locks the cassette in the ‘off’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. BFP, blue fluorescent protein; gcry1 , gamma crystallin 1 promoter; myl7 , cardiac myosin light chain 7 promoter; 2 A, porcine teschvirus-1 2A peptide; mRFP, monomeric red fluorescent protein; pA, transcription termination and polyadenylation signal; SA, splice acceptor.

Techniques: Plasmid Preparation, Marker, Cloning, CRISPR, In Vivo, Generated

Recovery of rbbp4 and rb1 UFlip floxed conditional alleles by GeneWeld CRISPR-Cas9 targeted integration. Table 2—source data 1. rbbp4 and rb1 UFlip embryo injection secondary marker F0 screening data and germline transmission data.

Journal: eLife

Article Title: Cre/ lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

doi: 10.7554/eLife.71478

Figure Lengend Snippet: Recovery of rbbp4 and rb1 UFlip floxed conditional alleles by GeneWeld CRISPR-Cas9 targeted integration. Table 2—source data 1. rbbp4 and rb1 UFlip embryo injection secondary marker F0 screening data and germline transmission data.

Techniques: CRISPR, Injection, Marker, Transmission Assay, Expressing

( A ) Diagram of rb1 gene model with exon 2 gRNA, the stop-PRISM-myl7:GFP targeting vector, and the resulting rb1-stop-myl7:GFP allele after GeneWeld CRISPR-Cas9 targeted integration. ( B ) F1 adult rb1-stop 5’ and 3’ junction analysis shows precise integration of the stop-PRISM cassette at the exon 2 target site. ( C ) Immunolocalization and quantification of pH3-labeled cells in control +/+ (n=3), rb1 stop/+ (n=3), rb1 stop/stop (n=3), and rb1 Δ7 / Δ7 (n=3) 5 dpf sectioned head tissue in the midbrain optic tectum and thalamic region (top row) and retina (bottom row) in both rb1 stop/stop and rb1 Δ7 / Δ7 homozygotes. rb1 stop/stop vs. +/+midbrain (**** p<0.0001) and retina (**** p<0.0001), rb1 stop/stop vs. rb1 Δ7 / Δ7 midbrain (n.s. p=0.2951) and retina (n.s. p=0.1534). Th, thalamic region; OT, optic tectum; R, retina. Error bars represent mean ± s.e.m. with two-tailed t -test. Scale bars: 50 μm. Figure 7—figure supplement 1—source data 1. Source data for quantification of pH3 positive cells in midbrain and retina of wildtype +/+, heterozygous rb1 stop/stop , homozygous rb1 stop/stop , and homozygous rb1 Δ7 / Δ7 5 dpf larva.

Journal: eLife

Article Title: Cre/ lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

doi: 10.7554/eLife.71478

Figure Lengend Snippet: ( A ) Diagram of rb1 gene model with exon 2 gRNA, the stop-PRISM-myl7:GFP targeting vector, and the resulting rb1-stop-myl7:GFP allele after GeneWeld CRISPR-Cas9 targeted integration. ( B ) F1 adult rb1-stop 5’ and 3’ junction analysis shows precise integration of the stop-PRISM cassette at the exon 2 target site. ( C ) Immunolocalization and quantification of pH3-labeled cells in control +/+ (n=3), rb1 stop/+ (n=3), rb1 stop/stop (n=3), and rb1 Δ7 / Δ7 (n=3) 5 dpf sectioned head tissue in the midbrain optic tectum and thalamic region (top row) and retina (bottom row) in both rb1 stop/stop and rb1 Δ7 / Δ7 homozygotes. rb1 stop/stop vs. +/+midbrain (**** p<0.0001) and retina (**** p<0.0001), rb1 stop/stop vs. rb1 Δ7 / Δ7 midbrain (n.s. p=0.2951) and retina (n.s. p=0.1534). Th, thalamic region; OT, optic tectum; R, retina. Error bars represent mean ± s.e.m. with two-tailed t -test. Scale bars: 50 μm. Figure 7—figure supplement 1—source data 1. Source data for quantification of pH3 positive cells in midbrain and retina of wildtype +/+, heterozygous rb1 stop/stop , homozygous rb1 stop/stop , and homozygous rb1 Δ7 / Δ7 5 dpf larva.

Techniques: Plasmid Preparation, CRISPR, Labeling, Control, Two Tailed Test

Journal: eLife

Article Title: Cre/ lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

doi: 10.7554/eLife.71478

Figure Lengend Snippet:

Techniques: Recombinant, Plasmid Preparation, In Vitro, Expressing, Sequencing, Cloning, TA Cloning, SYBR Green Assay, Software, CRISPR

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crispr edits ice software by synthego (Synthego Inc)

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1) Product Images from "Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production"

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