whole plasmid sequencing  (Plasmidsaurus)

Journal: Cell Genomics

Article Title: Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design

doi: 10.1016/j.xgen.2026.101190

Figure Lengend Snippet: Benchmarking predictions of CRISPR-Cas9ko guide behavior at putative off-target sites with GUIDE-seq data (A) The specificity-defining region (SDR) refers to positions 4–20 along a guide sequence and the PAM. (B) Left: off-target site counts indicate the total number of potential off-target sites across the 114 guides in the GUIDE-seq dataset, stratified by the maximum number of mismatches in the SDR tolerated in the off-target search. “Bulge” off-target sites represent those with 4+ mismatches in the SDR, yet this mismatch count can be reduced with a single-nucleotide shift within the sequence. Right: the fraction of each set of off-target sites as reported on the left in each range of activity rates, which are measured by the percentage of GUIDE-seq reads at the off-target site relative to the guide’s on-target site. (C) Association between CFD scores and activity at all predicted off-target sites for the 114 guides in the GUIDE-seq dataset. Off-target sites that exceed two mismatches in the SDR are excluded due to their low propensity for activity. Off-target sites are binned into groups of 37 by their CFD score, and those with at least 1% as many GUIDE-seq reads as the on-target site are classified as active. For each bin, the median CFD value indicates the predicted activity rate, and the fraction of active off-target sites indicates the experimental activity rate, yielding a Pearson correlation of 0.93. Horizontal error bars capture the minimum and maximum CFD score within each bin. See also .

Article Snippet: Purified plasmids were verified by restriction enzyme digest and whole plasmid sequencing through Plasmidsaurus.

Techniques: CRISPR, Sequencing, Activity Assay

Journal: Cell Genomics

Article Title: Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design

doi: 10.1016/j.xgen.2026.101190

Figure Lengend Snippet: Assessment of the Jacquere library screening performance (A) The Pearson correlation between biological replicates and across cell lines for the Jacquere library screened with the pRDA_734 vector. Correlation is calculated upon the log fold change in guide abundance at day 21 post-transduction relative to plasmid DNA (pDNA) abundance. (B) Receiver operating characteristic analysis of cell viability screening data for the Jacquere library (pRDA_734 vector) and Brunello library screened in A375 cells. False positive rates are determined by nonessential genes and plotted against the true positive rate, which is determined by essential genes. (C) Precision-recall curve to compare the classification of essential genes between Jacquere and Brunello screening data. (D) Stratified analysis of false positive rates and false negative rates in screening data of the Jacquere and Brunello libraries. Guide Z scores are calculated relative to the mean and standard deviation of intergenic control guides, and the plots demonstrate the percentage of guides targeting essential (left) or nonessential (right) genes that deplete at the Z score cutoff, as indicated on the x axis. (E) Rule set 3 sequence+target (Chen tracrRNA) on-target efficacy scores for guides selected by each library to target the essential genes that deplete in Jacquere but not Brunello A375 screening data, thus representing false negatives unique to the Brunello library. For this subset of genes, the guides selected for the Jacquere library feature a higher distribution of efficacy scores (Mann-Whitney two-sided U test; p < 0.001). (F) Aggregate CFD scores for guides selected by each library to target the nonessential genes that deplete in the Brunello but not Jacquere A375 screening data represent false positives unique to the Brunello library. For this subset of genes, the guides selected for the Jacuere library feature a lower distribution of Aggregrate CFD scores (Mann-Whitney two-sided U test; p < 0.001). (G) Comparison of off-target viability effects with the use of single-guide and dual-guide vectors. The A375 screening data of the Jacquere library described in this manuscript are compared to screening data of the Vienna-single and Vienna-double libraries presented in Lukasiak et al. The bar color indicates the number of constructs (out of three) targeting a nonessential gene that depletes relative to intergenic controls. See also .

Article Snippet: Purified plasmids were verified by restriction enzyme digest and whole plasmid sequencing through Plasmidsaurus.

Techniques: Library Screening, Plasmid Preparation, Transduction, Standard Deviation, Control, Sequencing, MANN-WHITNEY, Comparison, Construct