Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.

ABSTRACT: A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable var

SUBMITTER: Haussmann IU 

PROVIDER: S-EPMC11694751 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature