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Synthego Inc ice inference of crispr edits software
A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative <t>CRISPR-edited</t> Bhimkol plantlets with healthy shoots and roots, ready for hardening.
Ice Inference Of Crispr Edits Software, 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
https://www.bioz.com/product/inference+crispr+edits+(ice/ice+synthego/bio_rxiv__64898__2026__05__13__724745-111-3-10
Average 86 stars, based on 1 article reviews
ice inference of crispr edits software - by Bioz Stars, 2026-09
86/100 stars

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1) Product Images from "CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol"

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

Journal: bioRxiv

doi: 10.64898/2026.05.13.724745

A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative CRISPR-edited Bhimkol plantlets with healthy shoots and roots, ready for hardening.
Figure Legend Snippet: A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative CRISPR-edited Bhimkol plantlets with healthy shoots and roots, ready for hardening.

Techniques Used: Transformation Assay, Selection, CRISPR

CRISPR-edited Bhimkol plants transferred to the transgenic net house, A) 03_01, B) 03_05, C) 03_06, D) 03_17 and E) Wild Type (Control)
Figure Legend Snippet: CRISPR-edited Bhimkol plants transferred to the transgenic net house, A) 03_01, B) 03_05, C) 03_06, D) 03_17 and E) Wild Type (Control)

Techniques Used: CRISPR, Transgenic Assay, Control

Sequence analysis of the INO gene in CRISPR/Cas12a-edited lines. Amino acid sequences from lines 03_01, 03_05, 03_06, and 03_17 were aligned with the wild type sequence. Red asterisks indicate stop codons.
Figure Legend Snippet: Sequence analysis of the INO gene in CRISPR/Cas12a-edited lines. Amino acid sequences from lines 03_01, 03_05, 03_06, and 03_17 were aligned with the wild type sequence. Red asterisks indicate stop codons.

Techniques Used: Sequencing, CRISPR

Analysis of YABBY and HMG-box_2 domain in the WT and CRISPR-edited lines. The CRISPR-edited Bhimkol lines exhibit a complete absence of the HMG_box_2 and truncated YABBY domains.
Figure Legend Snippet: Analysis of YABBY and HMG-box_2 domain in the WT and CRISPR-edited lines. The CRISPR-edited Bhimkol lines exhibit a complete absence of the HMG_box_2 and truncated YABBY domains.

Techniques Used: CRISPR

Related Articles

CRISPR:

Article Title: Dual inhibition of intercellular adhesion molecule-1 and nucleolin reduces RSV infection efficiency in human respiratory organoids
Article Snippet: .. Analysis was done using inference of CRISPR edits (ICE) ( https://www.synthego.com/help/ice ) showed that the KO scores for ICAM-1-KO and EGFR-KO iPSCs were nearly 100 ( A and A). ..

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity.
Article Snippet: Successful PCR amplification was confirmed by agarose gel electrophoresis and PCR amplicons were transferred to ELIM Biopharm for Sanger sequencing. .. Editing efficiencies (Table S3) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout. ..

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol
Article Snippet: .. Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence. ..

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol
Article Snippet: .. Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively. ..

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells
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). .. Additionally, PCR products were digested with T7 Endonuclease I (T7EI; NEB M0302S) and analyzed by agarose gel electrophoresis to visualize indels.

Article Title: A mitochondria-driven quality control mechanism for peroxisomal membrane proteins.
Article Snippet: .. Editing efficiency was assessed using the Inference of CRISPR Edits (ICE) CRISPR Analysis Tool (Synthego Performance Analysis v3.0.). ..

Gene Knockout:

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity.
Article Snippet: Successful PCR amplification was confirmed by agarose gel electrophoresis and PCR amplicons were transferred to ELIM Biopharm for Sanger sequencing. .. Editing efficiencies (Table S3) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout. ..

Software:

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol
Article Snippet: .. Utilizing ICE (Inference of CRISPR Edits) software from Synthego provides a high level of bioinformatic confidence. ..

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol
Article Snippet: .. Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively. ..

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells
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). .. Additionally, PCR products were digested with T7 Endonuclease I (T7EI; NEB M0302S) and analyzed by agarose gel electrophoresis to visualize indels.

Sequencing:

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells
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). .. Additionally, PCR products were digested with T7 Endonuclease I (T7EI; NEB M0302S) and analyzed by agarose gel electrophoresis to visualize indels.

other:

Article Title: Optimized lentivirus-derived virus-like particles for efficient delivery of Cas9-based genome editors.
Article Snippet: Cells were collected and washed with 1 ml phosphate buffered saline (PBS), then stained with B2M-PE (clone 2M2, BioLegend, San Diego, CA), CD45-APC (clone HI30, BioLegend), CD44-PE-Cy7(clone IM7, BioLegend), CD29-APC (clone MAR4, BD Biosciences, WUborn, MA), CD47-FITC (clone miap301, BioLegend), and viability dye (Fixable Viability Dye eFluor 780, eBioscience San Diego, California) antibodies at 4◦C for 20 min at dilutions recommended by the manufacturer.



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A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative <t>CRISPR-edited</t> Bhimkol plantlets with healthy shoots and roots, ready for hardening.
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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
Inference Of Crispr Edits Ice, 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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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
Ice Inference Of Crispr Edits Tool, 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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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
Inference Of Crispr Edits Ice Algorithm, 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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a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following <t>CRISPR/Cas9</t> editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.
Ice Inference Of Crispr Edits Analyses, 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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(A) Schematic overview of experimental design for <t>CRISPR/Cas9</t> or cytosine base editing (CBE) mediated editing of HOTSCRAMBL at rs17437411 in human CD34 + HSPCs, followed by genotyping, RNA, and in vitro functional assays at indicated timepoints. (B and C) Representative flow cytometry plots of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC (CD34 + CD45RA-CD90 + CD133 + EPCR + ITGA3 + ) subsets at Day 5 post-editing in AAVS1 KO vs. HOTSCRAMBL KO (B), and AAVS1 CBE vs. HOTSCRAMBL rs17437411 (C). (D and E) Quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations at Day 5 (D) and Day 8 (E) post-editing based on flow cytometry. (F) Flow cytometric quantification of CD34 + CD45RA-CD90 + cells in S/G2/M phases at Day 5 by DyeCycle staining. (G) Representative CFSE flow cytometry plots showing cell division profiles of CD34 + CD45RA-CD90 + cells on Day 5. (H) Quantification of CFSE mean fluorescence intensity (MFI) in CD34 + CD45RA-CD90 + cells on Day 5. (I) Stacked bar plots of colony-forming unit (CFU) assay at 14 days post-plating, quantifying the distribution of erythroid (BFU-E), granulo-cyte/macrophage (CFU-G/M), and multilineage granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) colonies. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).
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Image Search Results


A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative CRISPR-edited Bhimkol plantlets with healthy shoots and roots, ready for hardening.

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: A) Induction of embryonic calli from male flowers of Bhimkol after 6 months of culture. B) A magnified view at 40x of embryogenic calli. C) Establishment of ECS after 8 months of culture was used for genetic transformation mediated through Agrobacterium harboring pSS03. D) Embryonic calli in selection medium with 40 mg/L hygromycin, and 250 mg/L timentin. E) A magnified view of putative transformed mature somatic embryos at 40x magnification. F) Shoots regeneration in M4 medium containing 40 mg/L hygromycin, and 250 mg/L timentin after one month of culture. G) Rooting from i n vitro regenerated shoots in the R1 rooting medium containing 40 mg/L hygromycin, and 250 mg/L timentin after 14 days of culture. H) Putative CRISPR-edited Bhimkol plantlets with healthy shoots and roots, ready for hardening.

Article Snippet: Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively.

Techniques: Transformation Assay, Selection, CRISPR

CRISPR-edited Bhimkol plants transferred to the transgenic net house, A) 03_01, B) 03_05, C) 03_06, D) 03_17 and E) Wild Type (Control)

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: CRISPR-edited Bhimkol plants transferred to the transgenic net house, A) 03_01, B) 03_05, C) 03_06, D) 03_17 and E) Wild Type (Control)

Article Snippet: Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively.

Techniques: CRISPR, Transgenic Assay, Control

Sequence analysis of the INO gene in CRISPR/Cas12a-edited lines. Amino acid sequences from lines 03_01, 03_05, 03_06, and 03_17 were aligned with the wild type sequence. Red asterisks indicate stop codons.

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: Sequence analysis of the INO gene in CRISPR/Cas12a-edited lines. Amino acid sequences from lines 03_01, 03_05, 03_06, and 03_17 were aligned with the wild type sequence. Red asterisks indicate stop codons.

Article Snippet: Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively.

Techniques: Sequencing, CRISPR

Analysis of YABBY and HMG-box_2 domain in the WT and CRISPR-edited lines. The CRISPR-edited Bhimkol lines exhibit a complete absence of the HMG_box_2 and truncated YABBY domains.

Journal: bioRxiv

Article Title: CRISPR/Cas12a-Mediated Knockout of the INNER NO OUTER ( INO ) Gene in Musa balbisiana cv. Bhimkol

doi: 10.64898/2026.05.13.724745

Figure Lengend Snippet: Analysis of YABBY and HMG-box_2 domain in the WT and CRISPR-edited lines. The CRISPR-edited Bhimkol lines exhibit a complete absence of the HMG_box_2 and truncated YABBY domains.

Article Snippet: Furthermore, analysis using ICE (Inference of CRISPR Edits) software from Synthego showed that lines 03_06 and 03_17 harboured 4-bp deletions within the target region at positions 97–100 and 96–99, respectively.

Techniques: CRISPR

a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following CRISPR/Cas9 editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following CRISPR/Cas9 editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.

Article Snippet: Sanger sequence traces were analyzed using the ICE (Inference of CRISPR Editing) deconvolution tool from Synthego. ( https://ice.synthego.com ; Synthego Performance Analysis, ICE Analysis.

Techniques: Sequencing, CRISPR, Expressing, Plasmid Preparation, Biomarker Discovery, Cell Culture, Imaging

(A) Schematic overview of experimental design for CRISPR/Cas9 or cytosine base editing (CBE) mediated editing of HOTSCRAMBL at rs17437411 in human CD34 + HSPCs, followed by genotyping, RNA, and in vitro functional assays at indicated timepoints. (B and C) Representative flow cytometry plots of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC (CD34 + CD45RA-CD90 + CD133 + EPCR + ITGA3 + ) subsets at Day 5 post-editing in AAVS1 KO vs. HOTSCRAMBL KO (B), and AAVS1 CBE vs. HOTSCRAMBL rs17437411 (C). (D and E) Quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations at Day 5 (D) and Day 8 (E) post-editing based on flow cytometry. (F) Flow cytometric quantification of CD34 + CD45RA-CD90 + cells in S/G2/M phases at Day 5 by DyeCycle staining. (G) Representative CFSE flow cytometry plots showing cell division profiles of CD34 + CD45RA-CD90 + cells on Day 5. (H) Quantification of CFSE mean fluorescence intensity (MFI) in CD34 + CD45RA-CD90 + cells on Day 5. (I) Stacked bar plots of colony-forming unit (CFU) assay at 14 days post-plating, quantifying the distribution of erythroid (BFU-E), granulo-cyte/macrophage (CFU-G/M), and multilineage granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) colonies. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Journal: bioRxiv

Article Title: Genetic variation reveals a homeotic long noncoding RNA that modulates human hematopoietic stem cells

doi: 10.1101/2025.07.16.664824

Figure Lengend Snippet: (A) Schematic overview of experimental design for CRISPR/Cas9 or cytosine base editing (CBE) mediated editing of HOTSCRAMBL at rs17437411 in human CD34 + HSPCs, followed by genotyping, RNA, and in vitro functional assays at indicated timepoints. (B and C) Representative flow cytometry plots of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC (CD34 + CD45RA-CD90 + CD133 + EPCR + ITGA3 + ) subsets at Day 5 post-editing in AAVS1 KO vs. HOTSCRAMBL KO (B), and AAVS1 CBE vs. HOTSCRAMBL rs17437411 (C). (D and E) Quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations at Day 5 (D) and Day 8 (E) post-editing based on flow cytometry. (F) Flow cytometric quantification of CD34 + CD45RA-CD90 + cells in S/G2/M phases at Day 5 by DyeCycle staining. (G) Representative CFSE flow cytometry plots showing cell division profiles of CD34 + CD45RA-CD90 + cells on Day 5. (H) Quantification of CFSE mean fluorescence intensity (MFI) in CD34 + CD45RA-CD90 + cells on Day 5. (I) Stacked bar plots of colony-forming unit (CFU) assay at 14 days post-plating, quantifying the distribution of erythroid (BFU-E), granulo-cyte/macrophage (CFU-G/M), and multilineage granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) colonies. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Article Snippet: CRISPR/Cas9 editing efficiency and frameshift composition were analyzed using the Inference of CRISPR Edits (ICE) tool ( https://ice.synthego.com/#/ ).

Techniques: CRISPR, In Vitro, Functional Assay, Flow Cytometry, Staining, Fluorescence, Colony-forming Unit Assay

(A) Experimental workflow for using Locked Nucleic Acid (LNA) GapmeR knockdown of HOTSCRAMBL in human CD34 + HSPCs (in AAVS1 CBE and HOTSCRAMBL rs17437411). Cells were treated with HOTSCRAMBL-targeting or control GapmeRs and analyzed by flow cytometry and CFU assay at Day 5. (B) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in cells treated with GapmeRs or control oligonucleotides under AAVS1 CBE or HOTSCRAMBL rs17437411 editing conditions at day 5, respectively. GR = GapmeR. (C) Stacked bar plots of CFU assay at 14 days post-plating, showing the distribution of BFU-E, CFU-G/M, and CFU-GEMM colonies. Cells were treated with GapmeRs or control oligonucleotides under AAVS1 CBE or HOTSCRAMBL rs17437411 editing conditions, respectively. (D) Experimental workflow for overexpression of HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs following CRISPR/Cas9-mediated HOTSCRAMBL knockout in human CD34 + HSPCs. Cells were transduced with SnoVectors expressing HOTSCRAMBL or HOTSCRAMBL rs17437411, and assessed by flow cytometry and CFU at Day 5. (E) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in cells overexpressed with HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs under AAVS1 KO or HOTSCRAMBL KO editing conditions at day 5, respectively. (F) Stacked bar plots of CFU assay at 14 days post-plating, showing the distribution of BFU-E, CFU-G/M, and CFU-GEMM colonies. Cells were overexpressed with HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs under AAVS1 KO or HOTSCRAMBL KO editing conditions at day 5, respectively. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Journal: bioRxiv

Article Title: Genetic variation reveals a homeotic long noncoding RNA that modulates human hematopoietic stem cells

doi: 10.1101/2025.07.16.664824

Figure Lengend Snippet: (A) Experimental workflow for using Locked Nucleic Acid (LNA) GapmeR knockdown of HOTSCRAMBL in human CD34 + HSPCs (in AAVS1 CBE and HOTSCRAMBL rs17437411). Cells were treated with HOTSCRAMBL-targeting or control GapmeRs and analyzed by flow cytometry and CFU assay at Day 5. (B) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in cells treated with GapmeRs or control oligonucleotides under AAVS1 CBE or HOTSCRAMBL rs17437411 editing conditions at day 5, respectively. GR = GapmeR. (C) Stacked bar plots of CFU assay at 14 days post-plating, showing the distribution of BFU-E, CFU-G/M, and CFU-GEMM colonies. Cells were treated with GapmeRs or control oligonucleotides under AAVS1 CBE or HOTSCRAMBL rs17437411 editing conditions, respectively. (D) Experimental workflow for overexpression of HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs following CRISPR/Cas9-mediated HOTSCRAMBL knockout in human CD34 + HSPCs. Cells were transduced with SnoVectors expressing HOTSCRAMBL or HOTSCRAMBL rs17437411, and assessed by flow cytometry and CFU at Day 5. (E) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in cells overexpressed with HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs under AAVS1 KO or HOTSCRAMBL KO editing conditions at day 5, respectively. (F) Stacked bar plots of CFU assay at 14 days post-plating, showing the distribution of BFU-E, CFU-G/M, and CFU-GEMM colonies. Cells were overexpressed with HOTSCRAMBL or HOTSCRAMBL rs17437411 RNAs under AAVS1 KO or HOTSCRAMBL KO editing conditions at day 5, respectively. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Article Snippet: CRISPR/Cas9 editing efficiency and frameshift composition were analyzed using the Inference of CRISPR Edits (ICE) tool ( https://ice.synthego.com/#/ ).

Techniques: Knockdown, Control, Flow Cytometry, Colony-forming Unit Assay, Over Expression, CRISPR, Knock-Out, Transduction, Expressing

(A) Experimental workflow for gene editing in human CD34 + HSPCs followed by sorting of CD34 + CD45RA-CD90 + populations and bulk RNA sequencing to assess differential expressed genes (DEGs) and splicing events. (B) Volcano plots showing DEGs in HOTSCRAMBL KO vs. AAVS1 KO (left panel) and HOTSCRAMBL rs17437411 vs. AAVS1 CBE (right panel). (C) Heatmap of HOXA cluster genes in AAVS1 KO, HOTSCRAMBL KO, AAVS1 CBE and HOTSCRAMBL rs17437411 conditions. Color bar represents z-score (row-normalized expression), while circle size reflects relative expression level (non-normalized). (D) Sashimi plots of read coverage tracks at the HOXA9 locus showing exon usage and splicing patterns. (E) Quantification of total splice junction read counts mapped to HOXA9 exon 1-2. (F) Bar plot of normalized HOXA9 transcript counts derived from mRNA sequencing. (G) Western blot analysis of HOXA9 protein expression (up panel) and quantification normalized to VINCULIN (bottom panel). (H) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in CD34 + HSPCs edited with AAVS1 -sgRNA (control) or two independent HOXA9 -targeting sgRNAs ( HOXA9 -sg1 and HOXA9 -sg2) using CRISPR/Cas9. (I) Schematic of lentiviral overexpression strategy using HOXA9 pre-mRNA or mature mRNA in AAVS1 CBE or HOTSCRAMBL rs17437411 edited groups, respectively. (J) Rescue of LT-HSC frequency in AAVS1 CBE or HOTSCRAMBL rs17437411 edited group by lentiviral expression of HOXA9 pre-mRNA or mRNA, quantified by flow cytometry. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Journal: bioRxiv

Article Title: Genetic variation reveals a homeotic long noncoding RNA that modulates human hematopoietic stem cells

doi: 10.1101/2025.07.16.664824

Figure Lengend Snippet: (A) Experimental workflow for gene editing in human CD34 + HSPCs followed by sorting of CD34 + CD45RA-CD90 + populations and bulk RNA sequencing to assess differential expressed genes (DEGs) and splicing events. (B) Volcano plots showing DEGs in HOTSCRAMBL KO vs. AAVS1 KO (left panel) and HOTSCRAMBL rs17437411 vs. AAVS1 CBE (right panel). (C) Heatmap of HOXA cluster genes in AAVS1 KO, HOTSCRAMBL KO, AAVS1 CBE and HOTSCRAMBL rs17437411 conditions. Color bar represents z-score (row-normalized expression), while circle size reflects relative expression level (non-normalized). (D) Sashimi plots of read coverage tracks at the HOXA9 locus showing exon usage and splicing patterns. (E) Quantification of total splice junction read counts mapped to HOXA9 exon 1-2. (F) Bar plot of normalized HOXA9 transcript counts derived from mRNA sequencing. (G) Western blot analysis of HOXA9 protein expression (up panel) and quantification normalized to VINCULIN (bottom panel). (H) Flow cytometric quantification of CD34 + CD45RA-, CD34 + CD45RA-CD90 + CD133 + , and LT-HSC populations in CD34 + HSPCs edited with AAVS1 -sgRNA (control) or two independent HOXA9 -targeting sgRNAs ( HOXA9 -sg1 and HOXA9 -sg2) using CRISPR/Cas9. (I) Schematic of lentiviral overexpression strategy using HOXA9 pre-mRNA or mature mRNA in AAVS1 CBE or HOTSCRAMBL rs17437411 edited groups, respectively. (J) Rescue of LT-HSC frequency in AAVS1 CBE or HOTSCRAMBL rs17437411 edited group by lentiviral expression of HOXA9 pre-mRNA or mRNA, quantified by flow cytometry. All data are presented as mean ± SD, significance is indicated as ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, or n.s. (not significant).

Article Snippet: CRISPR/Cas9 editing efficiency and frameshift composition were analyzed using the Inference of CRISPR Edits (ICE) tool ( https://ice.synthego.com/#/ ).

Techniques: RNA Sequencing, Expressing, Derivative Assay, Sequencing, Western Blot, Control, CRISPR, Over Expression, Flow Cytometry