sgRNA structural constraints and genetic limitations for efficient Cas9 genome editing to generate knock-outs

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. Here we describe constraints in sgRNA design originating from maintaining the secondary structure of the sgRNA that is essential for high- efficiency DNA scission of Cas9, but aberrant with about 50% of sequences adjacent to PAM sites in Drosophila. We developed an sgRNA design tool (PlatinumCRISPR) to evaluate base-pairing for optimal design of highly efficient sgRNAs for Cas9 genome editing. Applied to generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N6 methylated adenosines (m6A) in mRNA we further discovered, that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions, however, can be removed by standard genetic recombination and chromosome exchange.