sgRNA Design and Confirmation

CRISPR/Cas9 technologies inducing targeted mutations in Drosophila has been used widely. The mature CRIPSR/Cas9 adaptive immune system has been simplified to two components for use in genome engineering: Cas9 and a single chimeric RNA referred to as a chiRNA or single guide RNA (sgRNA). The specificity is determined by a 20 nt sequence at the end of the sgRNA, which can be altered to match any desired sequence in the DNA. Besides, it is the sgRNA taking Cas9 protein to the target sites.

CD BioSciences provides a series of sgRNA service, including sequence design, synthesis, validation of efficiency and off-target analysis. In the past years, our Drosophila experts have been focusing on the development of gene-editing technology and are following the latest technique in sgRNA design and efficiency analysis. We work closely as your partners to bring you satisfied products, and provide one-stop services ranging from sgRNA design/synthesis as long as quality analysis. Relying on proprietary gene synthesis platform, we have successfully completed efficient and high-throughput gene/genome editing.

What is sgRNA?

sgRNA generally ~100 nt is formed by hybridization of a tracrRNA and a crRNA. The targeting crRNA is composed by a ~20 nt sequence (the protospacer) complementary to the target DNA with the sequence requirement of a protospacer adjacent motif (PAM) (5'-NGG for the mostly used SpCas9, 5'-NNGRRT for saCas9). CRISPR systems from other bacterial species have different PAM requirements. tracerRNA and crRNA form a hybrid, and then the endogenous RNA enzyme III cleaves the crRNA-tracrRNA complex to form mature crRNA, which is combined into crRNA-tracrRNA-Cas9 functional unit. The difference in efficiency between plasmid and sgRNA design strategies may influence the success of CRISPR/Cas9 editing for the most part.

Fig.1 sgRNA in CRISPR/Cas9 system for genome editing (Gratz SJ et al. 2015)

sgRNA Design and Confirmation Service

Previously mentioned sgRNA is one of the most important part in gene editing action. The cutting efficiency of different sgRNA sites varies greatly, which may be caused by various reasons, such as the secondary structure of sgRNA and the thermal stability of sgRNA-DNA structure. To facilitate high-quality gRNA design, we offer a professional general CRISPR sgRNA strategy includes mutagenesis, insertion, deletion, incorporation, and desired modification to serve you well-workable designed products.

Service	Descriptions
sgRNA design	Design sgRNA based on clients' destination, whether insertion, deletion or others. Design sgRNA based on the specific of selected target edit regions on sequence analysis (at least 3 sgRNA)
sgRNA construction	Cloning gRNA into expression plasmids (most popular vector pCFD1-4, or clients can choose your own plasmid.) Purification of plasmid
sgRNA validation (optional)	Embryo-based Assay Microinjection of sgRNA into Act5c-Cas9 transgenic embryos, and screening for the efficiency of sgRNA by PCR. Cell-free Assay Cas9 protein and sgRNA are incubating with genome DNA in vitro. Then perform sequence analysis to this sample.

Why CD BioSciences?

We have record-proven expertise and experience in designing and generating sRNA for Drosophila CRISPR/Cas9 gene manipulation. We promise ~2 weeks fast turnaround sgRNA design service and comprehensive efficiency analysis with best after-sale, to be your faithful companion.

We support the delivery of sgRNA plasmids, or creation of stable sgRNA transgenic Drosophila lines by injecting sgRNA plasmids for further crossing with Cas9 transgenic flies.
We provide a comprehensive library of Drosophila sgRNA sequences and modification strategy targeting transcriptional expression of Drosophila sgRNA plasmids. In Drosophila, gRNAs are usually transcribed by the RNA polymerase III-dependent promoter of the U6 snRNA gene, which requires a 5' G nucleotide to achieve the best transcription efficiency. If the first nucleotide of the target gene is not G, we recommend to use a 19 nt complementary fragment and a 5' G.
We offer customized sgRNA site solutions for the characteristics of the Drosophila genome and clients' need. The generation of an indel scrambling reading frame and the usage of two gRNAs targeting the 5' and 3' ends of the gene can both introduce null mutations in the coding gene. The most straightforward solution is to generate an additional insert shortly after Initiation codon. Although the combination of two sgRNAs can technically achieve the deletion or replacement of large fragments, this event requires the efficiency and accuracy of the dual sgRNA. We recommend that sgRNAs should target the insertion site or close to it, and consider the distance effect between sites.

CD BioSciences can design sgRNA not just in the protein-coding region but also in RNA localization elements, ORF, promoter, and intronic regions. If you could not find what you really want in the website, please feel free to contact us.

Reference

Gratz SJ, et al. (2015). CRISPR-Cas9 Genome Editing in Drosophila. Curr Protoc Mol Biol. 111: 31.2.1-31.2.20.

For research use only. Not intended for any clinical use.

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