Journal: Journal of Biological Chemistry

Article Title: Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

doi: 10.1074/jbc.m110.146050

Figure Lengend Snippet: FIGURE 3. Engineering a full-length PHR1 cDNA. Indicated by thin black lines are products generated by 5-RACE, 3-RACE, and RT-PCR that were used to form the full-length PHR1 cDNA. An N-terminal cassette was formed by joining products A and B using a NotI site. A C-terminal cassette was formed by joining products G, H, and F using SphI and EagI. The N- and C-terminal cassettes were joined to form the full-length PHR1 cDNA using a unique (*) SmaI site.

Article Snippet: 3 -RACE—Poly(A)RNA fromChlamydomonas strain cc-125 was reverse-transcribed using cDNA cloning primer (Integrated DNA Technologies) and ImProm-II reverse transcriptase (Promega).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) The workflow takes linear DNA library as input. ( b ) Ribosome display links genotype (RNAs transcribed from DNA input library that are stop codon free, and stall ribosome at the end of the transcript) and phenotype (folded VHH protein tethered to ribosomes due to the lack of stop codon in the RNA). ( c ) Selection cycle that enriches DNA encoding for VHHs that binds immobilized targets. ( d ) High throughput sequencing of full-length VHHs. ( e ) Sequences are grouped into clusters based on similarity of their CDRs, each cluster is distinct and represent a unique binding family. ( f ) The system outputs one representative sequence from each cluster to be synthesized and characterized for specific downstream applications. ( g ) Workflow for generating VHH library. VHH CDR randomization was introduced by PCR using a hairpin oligo (blocks DNA end from ligation) and an oligo with random 5’ sequence, followed by orientation-controlled ligation. Three successive PCR plus ligation cycles randomizes all three CDRs. ( h ) The final DNA library sequence structure. ( i ) One round of ribosome display and anti-Myc selection was performed after randomization of CDR1 and CDR2. The pie chart shows percentage of indicated sequence categories before and after anti-Myc selection. ( j ) Length distribution of DNA region encoding CDR1 of the VHH library before and after anti-Myc selection. Arrows indicate all correct-frame lengths showing increased percentage after anti-Myc selection.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Selection, Next-Generation Sequencing, Binding Assay, Sequencing, Synthesized, Ligation

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Position-wise amino acid profile of natural VHHs (298 VHHs, PDB) and ( b ) synthetic VHHs. Amino acids were color coded according to labels to the right, B indicates an empty position. Bar height is the relative percentage of each amino acids. The two most common amino acids were shown as patterned bars while others were shown as solid bars. ( c ) Plot of diversity index (as 1 – Gini index) for each amino acid position of natural VHHs and ( d ) synthetic VHHs.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques:

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Amino acid sequences encoded by frames that serve as templates for VHH library generation were aligned to the corresponding segments of the human IGHV3-23 (hIGHV3-23) or IGHJ4 (hIGHJ4). Positions in hIGHV3-23/hIGHJ4 that are identical to the corresponding position in at least one VHH frames are highlighted in orange. Positions in VHH frames that are identical to the corresponding position in hIGHV3-23/hIGHJ4 are highlighted in orange. hIGHV3-23 positions not identical to any VHH frames are numbered according to its position within the segment. Asterisks indicate VHH hallmark residues thought to be required for VHH’s independence of light chain. ( b ) Percent homology of VHH frames to the closest human gene. ( c ) List of VHH residues at positions numbered in (a) and representative human IGHV genes that encode the same VHH residue at the corresponding position. None: no human IGHV genes has the VHH residue at the corresponding position.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Residue

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Immobilization strategy for the target proteins: 3xFlag-tagged EGFP or RBD. ( b ) Pair-wise CDR match score (based on BLOSUM62 matrix) were calculated for 2000 randomly selected sequences from input library and output libraries after 3 rounds of selection. High match score populations appeared in the output libraries. Combining CDR1 and 2 match scores further separated high and low score population and a match score of 35 (black dashed line) was chosen as cut-off for downstream clustering analysis. ( c ) Percentage of indicated sequence categories in the input library and output libraries (EGFP, RBD). ( d ) Number of unique and shared clusters identified in EGFP and RBD output libraries. ( e ) Number of sequences for each size of RBD unique clusters. ( f ) ELISA assay revealed 3 strong binders (“s”) to RBD, 7 weak binders (“w”) and ( g ) 4 non-binders (“n”) among the 14 VHHs chosen for characterization. ( h ) SARS-CoV-2 S pseudotyped lentivirus neutralization assay showed 6 VHHs inhibiting infection >30% at 1μM on HEK293T expressing ACE2 and TMPRSS2. Data shown are two technical replicates, bars indicate the average of data, circles indicate values of each replicate.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Selection, Sequencing, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Expressing

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Amino acid profile of representative VHH sequence for each unique cluster identified from RBD and EGFP output libraries (“output binders”, 932 sequences). Plotted as described in . ( b ) Plot of diversity index (as 1 – Gini index) for each amino acid position of output binder VHHs.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Sequencing

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) r 2 values for the amino acid percentages in the indicated sequence group pairs at each CDR position. 298 natural VHHs (natural) and 298 randomly sampled sequences from input library (input) and output binders (output) were analyzed. Three random sampling trials were performed to generate three r 2 for each position. ( b ) Scatter plots of the percentage of each amino acid in the input library and the output binders and ( c ) that in the natural VHHs and the output binders at representative CDR positions. Circles are the mean and error bars are the standard deviation of data. ( d ) Root mean square error (RMSE, relative to Y = X line) values for the indicated sequence group pairs at each CDR position. Using the same randomly sampled sequences as (a). ( e ) Plot showing the similarity distances between the three sequence groups, with each connecting line length between two sequence groups indicating their RMSE. Vertical dashed lines indicate the middle point of the distance between output and natural sequence groups

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Sequencing, Sampling, Standard Deviation

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Affinity maturation workflow. ( b ) Two representative sections of position-wise post-minus pre-affinity maturation amino acid percent point change profile. White values indicate the original amino acid, yellow values indicate the beneficial mutation. Empty positions indicate amino acids not detected in either the pre-or post-selection libraries. ( c ) ELISA assay of VHH variants. ( d ) SARS-CoV-2 S pseudotyped lentivirus neutralization assay of VHHs on HEK293T expressing ACE2 and TMPRSS2. For (c) and (d), data shown are two technical replicates, bars indicate the average of data, circles indicate values of each replicate. ( e ) Scatter plot of ELISA assay absorbance versus pseudotyped lentivirus neutralization as percent infection inhibited. VHH concentration for both assays were 50 nM. Values are average of two technical replicates. Numbers on linear fitting lines were r 2 value for data within each family. ( f ) Dose-response curve for neutralization of pseudotyped lentiviral infection by VHHs. Markers are average of three technical replicates, error bars are standard deviation. ( g ) IC50 calculated from data in (f), presented as mean ± standard deviation.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Mutagenesis, Selection, Enzyme-linked Immunosorbent Assay, Neutralization, Expressing, Infection, Concentration Assay, Standard Deviation

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , b , Schematic of genome-wide CRISPR off-target detection using MRE11 ChIP–seq. a , Cells are unsynchronized, so only some cells have MRE11 at Cas9 cut sites at a given time (DISCOVER-Seq). b , Inhibition of NHEJ directs DNA repair to slower, MRE11-dependent pathways. c , Effect of repair factor inhibition on MRE11 residence at the VEGFA site 3 on-target site, measured by ChIP–qPCR estimating ‘reads per million’ (RPM) enrichment at 12 h after Cas9 delivery in HEK293T cells. Each point corresponds to a different biologically independent replicate of a sample exposed to the DNA repair inhibitor listed in the x axis. Red line is the mean of two biologically independent replicates. Samples with DNA-PKcs inhibition ( n = 4) have significantly higher estimated RPM compared with samples without DNA-PKcs inhibition ( n = 8) using two-sided Student’s t -test ( P = 9.65 × 10 −5 ). d , Increased MRE11 residence upon DNA-PKcs inhibition using Ku-60648 (red) versus without inhibition (blue), measured by ChIP–qPCR. Measured over multiple time points (4 h, 12 h, 24 h) after delivery of Cas9 targeting VEGFA site 2 in HEK293T (left plot), with Cas9 targeting FANCF site 2 in K562 at 12 h (middle plot) and with Cas9 targeting HEK site 4 in HEK293T at 12 h (right plot). Plots display the mean over two biologically independent replicates for left and middle plots, and one biologically independent replicate for the right plot. e , Plot of estimated RPM enrichment normalized to the no drug sample from data in panel d , for sample pairs with (‘Ku-60648’) or without (‘no drug’) DNA-PKcs inhibition. Normalized RPM enrichment with DNA-PKcs inhibition was significantly higher than without inhibitor ( P = 0.0001), using two-sided Wilcoxon signed-rank test. Red line indicates mean of n = 14 total samples pooled from panel d ; green points are HEK293T, VEGFA site 2 ; purple points are HEK293T, HEK site 4 ; red points are K562, FANCF site 2 . *** P < 0.001.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Genome Wide, CRISPR, ChIP-sequencing, Inhibition, ChIP-qPCR

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , The proportion of 53BP1 foci relative to BRCA1 as detected by STED in cells exposed to Cas9 targeting a multi-target gRNA with 126 genome-wide target sites. N = 98 cells examined over four independent experiments, P = 0.00018 using two-sided Wilcoxon rank-sum test. b , The number of repair foci (53BP1 or BRCA1) as detected by STED in cells with or without Cas9 (‘+Cas9’ or ‘−Cas9’, respectively), with or without Ku-60648 (‘KU’ versus ‘nD’, respectively), targeting 126 genome-wide sites with a multi-target gRNA. N = 98 cells from four biologically independent replicates, P = 4.44 × 10 −10 using two-sided Wilcoxon rank-sum test between ‘−Cas9’ and ‘+Cas9’. Difference in no. of foci in each group was not significant (left, P = 0.15; right, P = 0.95). c , d , Representative images for panel b , ( c ) with Cas9 or ( d ) without Cas9. Red labels 53BP1, green labels BRCA1. Scale bar, 5 μm. e , Histogram of indels or no mutations (‘0’) at 48 h after Cas9 editing of ACTB , either without DNA-PKcs inhibitor (‘Cas9, no inhibitor’) or with inhibitor (‘Cas9, Ku-60648’). Untreated cells not exposed to Cas9 shown for reference (‘Untreated (no Cas9)’). Red bar displays mean over two biologically independent replicates. *** P < 0.001. NS, not significant.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Genome Wide

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , b , Plots of MRE11 ChIP–seq enrichment (number of reads within a 1.5-kb window centered at the cut site, per 1 million total reads, that is, RPM) for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition at all FANCF site 2 ( a ) or VEGFA site 2 ( b ) Cas9 target sites in K562 detected from the DNA-PKcs inhibited samples. Each point in the plot (15 in panel a , 178 in panel b ) corresponds to a putative target site. Significant differences ( P = 0.00081 or P = 4.57 × 10 −26 ) between y -axis and x -axis values were determined using two-sided Wilcoxon signed-rank test. c , d , Genome browser visualization of MRE11 enrichment at an on-target ( c ) and representative off-target ( d ) position from K562 with Cas9 targeting FANCF site 2 , with (red) or without (blue) DNA-PKcs inhibition. e , f , Plots of MRE11 ChIP–seq enrichment for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition at positions 10 kb downstream from the actual ( e ) FANCF site 2 or ( f ) VEGFA site 2 cut sites, to measure background enrichment adjacent to cut sites. MRE11 enrichment with ( y axis) versus without ( x axis) DNA-PKcs inhibition at the adjacent background locations was not significantly different ( P = 0.21 or 0.18), determined using two-sided Wilcoxon signed-rank test. g , Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 2 . Quantification of Extended Data Fig. . h , i , Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 3, FANCF site 2 ( h ) and HEK site 4 ( i ) gRNAs. Quantification of Extended Data Fig. . j , Venn diagram illustrating overlap in the identity of Cas9 target sites discovered from samples with DNA-PKcs inhibition (‘DNA-PKi only’; light blue), without DNA-PKcs inhibition (‘no drug only’; light yellow) or found in both samples (‘both’; light green). Four gRNAs were evaluated.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: ChIP-sequencing, Inhibition

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , Illustration of MRE11 ChIP-seq enrichment at a specific target site, visualized as a histogram of base pair coverage from sequencing reads along the genome. Only the two ends of each DNA fragment are sequenced. b , Genome browser visualization of MRE11 enrichment at the on-target site in K562 cells with Cas9 targeting FANCF site 2 . Y-axis is in log 10 scale. Overlaying red and blue graphs correspond to with or without DNA-PKcs, respectively. Black graph corresponds to samples without Cas9.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: ChIP-sequencing, Sequencing

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in K562 cells. b , FANCF site 2, VEGFA site 3, or HEK site 4 Cas9 target sites detected using DISCOVER-Seq versus DISCOVER-Seq+ in K562 or HEK293T cells, at 4 h, 12 h, or 24 h after Cas9 delivery. Source numerical data are available in source data.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques:

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a-b , Plot of the total number of initial off-target sites, for samples (a) without inhibitor and (b) with Ku-60648. Sites labeled as false positive, because they were also reported in corresponding negative control samples without Cas9, are colored red with the number of sites indicated above each bar. For x-axis, first row is cell type, second row is gRNA target (Vs2: VEGFA site 2, Hs4: HEK site 4, Vs3: VEGFA site 3, Fs2: FANCF site 2). c , Plot of MRE11 ChIP-seq enrichment at all DISCOVER-Seq+ detected target sites within a 1.5 kb window for Cas9 targeting VEGFA site 2 in WTC-11 iPSCs. Each point in the plot (55 total) corresponds to a putative target site. MRE11 enrichment from DISCOVER-Seq+ data (y-axis) versus DISCOVER-Seq data (x-axis) was significantly different (p = 1.24E-8), determined using the two-sided Wilcoxon signed-rank test. d-e , Venn diagram illustrating overlap in the VEGFA site 2 sites identified by (d) DISCOVER-Seq or (e) DISCOVER-Seq+ in K562 cells (blue), HEK293T cells (yellow), and WTC-11 iPSCs (purple). f-g , (f) Flow cytometry contour plots of T cell populations at day 7 following CRISPR editing with Cas9 (left) or Cas12a (right). X-axis gates by T cell binding to a phycoerythrin (PE)-conjugated HLA-A*02:p53 R175H peptide tetramer complex specific for the tgTCR. Y-axis gates by allophycocyanin (APC)-conjugated antibody against NGFR (CD271), introduced as part of the CRISPR HDRT as an editing control. Cells positive for both markers represent successful cancer-specific tgTCR integration. 9.7% of T cells are positive for both markers using Cas9 compared to 8.4% with Cas12a. (g) Same, but for negative control samples without gRNA. h , Gating strategy for flow cytometric analysis of live single T cells, used for panels f-g . i , Same as panel c , for Cas9 targeting PCSK9 in the liver of mice, with 30 total target sites. MRE11 enrichment from DISCOVER-Seq+ was significantly different (p = 7.90E-6), using the two-sided Wilcoxon signed-rank test. Source numerical data are available in source data.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Labeling, Negative Control, ChIP-sequencing, Flow Cytometry, CRISPR, Binding Assay, Control

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , For the 15 target sites (1 on-target, 14 off-targets) of the FANCF site 2 gRNA identified by DISCOVER-Seq+ (‘DSeq+’), the chart shows which sites are also identified by DISCOVER-Seq alone (‘+’ under ‘DSeq’), which sites have indels detectable by targeted deep sequencing (‘+’ under ‘Indels’) and which sites were also detectable by GUIDE-seq (‘+’ under ‘Gseq’). Target sites labeled with ‘N/A’ under ‘Indels’ were unable to be successfully amplified by PCR for targeted sequencing. b , Left plot, measurement of indels at the on-target and sole off-target site (OFF0) discovered by the original DISCOVER-Seq, for K562 cells with the FANCF site 2 gRNA, with or without Cas9 (‘+Cas9’ or ‘−Cas9’, respectively). Right plot, measurement of indels at off-target sites exclusively discovered by DISCOVER-Seq+. Plots display the mean of three biologically independent replicates; error bars represent ±1 s.d. from mean. * P < 0.05, ** P < 0.01 and *** P < 0.001, using two-sided Student’s t -test (exact P values in the for this figure). c , Venn diagram illustrating overlap in the identity of VEGFA site 2 and FANCF site 2 target sites identified by DISCOVER-Seq+ versus GUIDE-seq.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Sequencing, Labeling, Amplification

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in WTC-11 iPSCs. b , c , Genome browser visualization of MRE11 enrichment at an on-target ( b ) and representative off-target ( c ) position with four mismatches (‘4 mm’) in WTC-11 iPSCs with Cas9 targeting VEGFA site 2 . DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). d , Schematic of the DISCOVER-Seq+ protocol in the knock-in of a cancer neoantigen-specific tgTCR into the TRA locus of primary human T cells. e , TRA Cas9 target sites in primary T cells detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right). f , Genome browser visualization of MRE11 enrichment at a representative four-mismatch (‘4 mm’) off-target position in primary human T cells with Cas9 targeting TRA for knock-in of a tgTCR template. DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). g , Same as panel f , at another four-mismatch off-target position. h , Plot of MRE11 ChIP–seq RPM enrichment within a 1.5-kb window for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition, at all TRA Cas9 off-target sites in primary human T cells from the DNA-PKcs inhibited samples. Each point in the plot (20 total) corresponds to a putative target site. Differences ( P = 1 × 10 −3 or P = 0.69) between y -axis and x -axis values were determined using two-sided Wilcoxon signed-rank test. i , Same as panel h , for cells delivered with Cas9 but without gRNA (negative control). j , TRA Cas12a (Cpf1) target sites in primary T cells.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Knock-In, ChIP-sequencing, Inhibition, Negative Control

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , Schematic of DISCOVER-Seq+ protocol in mice. b – d , Genome browser visualization of MRE11 enrichment at the ( b ) PCSK9 on-target site (‘ON-target’), ( c ) one off-target site (‘OFF-target A’) and ( d ) another off-target site (‘OFF-target B’) with two mismatches each (‘2 mm’), in the liver of mice transduced with adenovirus expressing Cas9 targeting PCSK9 . Mice were dosed twice a day (b.i.d.) with either 25 mg kg −1 Ku-60648 (‘Ku-60648’; red) or with vehicle (‘no drug’; blue). e , Number of detected genome-wide target sites in the mouse genome mm10 with Cas9/gRNA targeting PCSK9 , identified using DISCOVER-Seq (DSeq) versus DISCOVER-Seq+ (DSeq+). N = 5 biologically independent replicates (mice) were used for each condition; two-sided Student’s t -test was used ( P = 0.0079). f , Venn diagram illustrating overlap in the mouse PCSK9 target sites identified by in vivo DISCOVER-Seq+ in this work, ‘DSeq+ (this work)’ (blue); by in vivo DISCOVER-Seq in this work, ‘DSeq (this work)’ (yellow); and in the original DISCOVER-Seq manuscript, ‘DSeq (Wienert et al. )’ (purple). All the target sites identified by ‘DSeq (this work)’ are also found in the other two groups. g , PCSK9 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in mouse liver. Off-target detection for each condition was performed on sequencing data pooled across five biologically independent mouse replicates. ** P < 0.01.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Transduction, Expressing, Genome Wide, In Vivo, Sequencing