Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

Abstract

Summary The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, sgRNA parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for the first time a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair, and achieved an overall mutagenesis rate significantly higher than previously reported. Our work presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…sgRNA2 could be considered more efficient than sgRNA1 in 5 of the 7 algorithms used. Moreover, sgRNA2 showed 5 G or C in the final 6 bp of the sgRNA that has been shown to facilitate cleavage (Ren et al ., 2014). Another possibility could be a specific profile of expression for the sgRNA#1, which is under the control of a Camelina U3 promoter (sgRNA#2 is under the control of a Camelina U6 promoter).…”

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

“…Indeed, the presence of the 39 GG motif in the protospacer is a better predictor of editing outcome than the algorithms derived from mammalian cells (Table S2) (Doench et al 2014). The overlap of the 39 GG dinucleotide in our guide design with the 39 G and/or two 39 purines of any arrangement in successful mammalian-cell guides (Doench et al 2014;Wang et al 2014;Wu et al 2014) and with the high GC content for the six 39 nucleotides of successful Drosophila guides (Ren et al 2014) makes it likely that the two key principles to emerge from our studies will apply to diverse species and cell lines.…”

“…Analysis of guide RNAs found to be effective in zebrafish (Gagnon et al 2014), Drosophila (Ren et al 2014), and mammalian cell lines (Doench et al 2014;Wang et al 2014;Wu et al 2014) suggests that the partial success of some 39 GG-shift guides in C. elegans might be due either to a G at position 20 of the target-specific sequences of the guide or to any combination of two purines at positions 19 and 20. Although those features are positively correlated with the effectiveness of guides in other species and might contribute to the partial success of guides in C. elegans, they cannot account for all the success of either the 39 GG guides or 39 GG-shift guides.…”

Immunohistochemistry Evaluation of TGF-β1, SOX-9, Type II Collagen and Aggrecan in Cartilage Lesions Treated with Conditioned Medium of Umbilical Cord Mesencyhmal Stem Cells in Wistar Mice (Rattus novergicus)

“…Moreover, this is the predominant type of collagen in cartilage. The extracellular matrix is also comprised of other specific collagens such as type IX and XI [17]. Following the increase in SOX-9 mRNA expression, the production of type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) is observed [8].…”