Figure 1 | Scientific Reports

Figure 1

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

Figure 1

Workflow for CRISPR gRNA design. Whole blood was collected from a total of 269 HIV-1-infected samples from 168 patients enrolled in the Drexel CARES Cohort. Genomic DNA was isolated from PBMCs and a two round, nested PCR amplified the HIV-1 LTR as described in the Methods. These LTR amplicons were then deep-sequenced. The resulting sequence was then examined as follows for gRNA design: (1) The training set (100 samples) was scanned for all possible 20-mer protospacers; (2) an off-target search filtered out potentially dangerous gRNAs present in the human genome; (3) all remaining protospacers were evaluated against the training dataset to; (4) rank all gRNAs by their in silico efficiency; (5) package the top ranking gRNAs; and (6) validate the selected gRNAs against a held-out testing set (169 samples).

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