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.

Service 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 will deliver up to 20ug of customized ready-to-use sgRNA
  • Convenience: ready-to-use sgRNA can be directly inject into animals or transfected into cells, improving the efficiency of gene editing experiments

Case Study – Synbio Technologies:

Synbio Technologies has designed 8 sgRNAs targeting several genes in mouse, and performed in vitro transfection. The experimental period was shortened to 2 days, and the sgRNA amount was increased to 10-20 ug. This change could mean a significant jump in efficiency for synthetic biology experiments utilizing CRISPR-Cas9.

Workflow:

sgRNA

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

Result:

  1. Clone DNA template into pUC57 vector, the sequencing result (Fig. 1) coincided with the designed sequence.

result1

Fig. 1. Comparison between blunt end ligation result and designed sequence

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

result2

Fig. 2. agarose gel electrophoresis of sgRNA

  1. 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.

result3

Fig. 3. sgRNA sequence verification using agarose gel electrophoresis

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

Service Specifications:


Service Name Product/Service Specifications Turnaround Time
(business day)
Deliverables Price
Ready-to-use sgRNA synthesis sgRNA design
DNA template synthesis
In vitro sgRNA transcription and purification
<10 sgRNA, 5
10-20 sgRNA, 10
>20 sgRNA,Inquiry
sgRNA
COA
Inquiry
Syno® negative control sgRNA Negative control sgRNA
In vitro sgRNA transcription and purification
5days Syno®-negative control sgRNA1
Syno®-negative control sgRNA2
Syno®-negative control sgRNA3
Inquiry

How to order


Tel
+1 732-230-3003

Email
service@synbio-tech.com

Fax
+1 609 228 5911

Online Inquiry
online inquiry submission form

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.