Journal: Scientific reports

Article Title: Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients.

doi: 10.1038/s41598-019-52353-9

Figure Lengend Snippet: Figure 6. D-LTR-268145 can account for the genetic diversity of the vQS from patient-derived subtype B HIV sequences better than a previously published gRNA in a high sensitivity in vitro CRISPR/Cas9 cleavage assay. (A) LTR clones from Drexel CARES Cohort patients were amplified from PBMC genomic DNA, cloned, and sequenced. Mismatches between the D-LTR-268145 and T-LTR-237050 gRNA and the patient LTR target sites were aligned. The activity score indicated the predicted likelihood that the gRNA would cleave the target sequences with that particular mismatch or combination of mismatches. (B) In vitro CRISPR/Cas9 cleavage results of patient-derived HIV sequences representing the vQS. The number of clones in the vQS indicates the number of individual plasmids that were mixed in equal ratios. For example, patient sample A107 had 6 plasmid clones to make the vQS (clone A107-5, A107-13, A107-15, A107-16, A107-52 and A107-65) and mismatches within the target site for each clone was represented in subpanel A. Statistical significance was determined using Kolmogorov–Smirnov test and an * indicated p-values <0.05. (C) The scatter plot represents the correlation of the observed percent cleaved in the in vitro assay shown in B versus the predicted cleavage from the MIT activity score.

Article Snippet: All gRNA expression plasmids (Catalog number 53186, 53187, 53188 and 53189) and Cas9 (Catalog number 41815) were purchased from Addgene. gRNA cloning was performed as previously described with some modifications62.

Techniques: Derivative Assay, In Vitro, CRISPR, Cleavage Assay, Clone Assay, Amplification, Activity Assay, Plasmid Preparation