Ready-to-use CRISPR Cas9 sgRNA Synthesis

CRISPR-Cas9 is one of the most convenient types of gene editing technology, and has been widely used in editing genes of many species[1]. CRISPR-Cas9 generally involves construction a plasmid containing the specific endogenous promoter and connection of the Cas9 gene and a synthetic sgRNA, followed by transfer into the cell or animal. However, plasmid construction and verification are both tedious and time-consuming. The degradation of the plasmid is relatively slow, which may prove to be inconvenient for subsequent experiments.

To improve the efficiency of the CRISPR-Cas9 system, Synbio Technologies has developed ready-to-use sgRNA synthesis services. We can perform sgRNA target design, DNA template synthesis of sgRNA, sgRNA in vitro transcription, and sgRNA purification, to provide customers ready-to-use sgRNA that can be directly transfected into cells or animals. Synbio Technologies’s ready-to-use sgRNA saves time on plasmid construction, and avoids the drawbacks of potentially non-degraded plasmids.

According to the publications, in vitro transcription of sgRNA has successfully edited the genes of different species including zebrafish[2], mouse[3]as well as filamentous fungi[4]etc. Synbio Technologies has also designed 3 universal negative control sgRNA for Human and Rat genome: Syno®-negative controls sgRNA1, sgRNA2, and sgRNA3. These sequences are used as a negative control in Human and Rat gene/genome editing experiments.

Competitive Advantages

  • One-stop solution: Synbio Technologies provides integrated services from sgRNA target design to high purity ready-to-use sgRNA production.
  • Fast delivery: In just 3 business days, Synbio Technologies will deliver up to 20μg of customized ready-to-use sgRNA
  • Convenience: ready-to-use sgRNA can be directly injected into animals or transfected into cells, improving the efficiency of gene editing experiments

Case Study

Synbio Technologies has designed a number of sgRNAs to target several genes in mouse, and performing in vitro transfection. The experimental period was shortened to 2 days, and the sgRNA amount was increased to 10-20 μg. This change could means a significant jump in efficiency for synthetic biology experiments utilizing CRISPR-Cas9.

Workflow

One-step synthesis of DNA template
One-step synthesis of DNA template

Results

  1. Clone DNA template into pUC57 vector, the sequencing result (Fig. 1) coincided with the designed sequence.
Fig. 1. Comparison between blunt end ligation result and designed sequence

2. Agarose gel electrophoresis of sgRNA obtained by in vitro transcription, clear bands shown in Fig. 2.

Fig. 2. agarose gel electrophoresis of sgRNA

3. sgRNA verification: Transcript sgRNA into cDNA, design sgRNA amplification primer, and obtain the complementary DNA sequence by PCR reaction. Clone DNA sequence into pUC57 vector; sequencing result (Fig. 3) showed the sgRNA sequence is correct.

*The template of Lane 1 is reverse transcripted cDNA, The template of Lane 2 is sgRNA digested by DNase I; The template of Lane 3 is in vitro transcripted DNA.

Fig. 3. sgRNA sequence verification using agarose gel electrophoresis

Service Specifications

References

[1]Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science. 2012,337(6096):816-821.
[2]Xiao A, Wang Z, Hu Y, et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish [J]. Nucleic Acids Res. 2013,41(14):e141.
[3]Fujii W, Kawasaki K, Sugiura K, Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease [J]. Nucleic Acids Res. 2013,41(20):e187.
[4]Liu R, Chen L, Jiang Y, Zhou Z, Zou G. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discovery. 2015.7.