crispr guide rna construct assembly

overview

CRISPR technology allows you to edit your target genome using a short, target-specific guide RNA (gRNA) and a specialized endonuclease. For target specificity, a properly designed guide sequence must be integrated into an empty gRNA vector in order to express the proper gRNA. This is your first step in preparing your gene editing experiment.

crispr-workflow

This workflow takes an empty CRISPR vector of your choice along with oligos based on your guide sequence and delivers final assemblies - ready for you to transform - 96 at a time.

Methodology

The destination vector provided by the customer is isolated as indicated and digested with the desired Type IIS restriction enzyme (NEB). Next, the restriction enzyme is heat inactivated, and the reaction mixture is cleaned up to remove small DNA fragments and residual enzyme. The resulting linearized vector is left with mutually non-compatible sticky ends (Figure 1). DNA is then stored until final ligation.

Figure 1  Digested vector with sticky ends.

Figure 1  Digested vector with sticky ends.

In parallel, 96 pairs of complementary oligos designed by the customer are ordered, resuspended in MilliQ water, and diluted to a 1uM working stock. To anneal, sense and antisense oligos are combined in a solution of T4 ligase buffer (NEB), heated to 95°C, and slowly cooled down to 4°C. The short dsDNA inserts that are produced contain overhangs that are compatible with the sticky ends from the linearized vector (Figure 2).

Figure 2  Short insert with overhangs compatible with the linearized vector’s sticky ends.

Figure 2  Short insert with overhangs compatible with the linearized vector’s sticky ends.

Finally, a mixture of T4 DNA ligase (NEB), linearized vector, and dsDNA insert is prepared for each construct design and incubated at room temperature to assemble the final product.

validation

To validate this Transcriptic protocol, 96 constructs were assembled as described, and 10 final assemblies were chosen at random for testing. Dh5α competent cells (Zymo Research Corporation) were transformed with each of these assembled constructs and grown for 18 hours on selective media. Finally, for each transformation, three colonies were randomly picked and tested using rolling circle amplification followed by Sanger sequencing. For every assembly tested, at least 2 out of 3 picked colonies had the expected sequence, with an average efficiency of 80% (Figure 3). Overall, 10 out of 10 constructs assembled correctly - yielding a 100% success rate.

Figure 3  Pie chart summarizing observed cloning efficiencies.

Figure 3  Pie chart summarizing observed cloning efficiencies.

conclusion

Transcriptic’s automated platform provides a robust, high-throughput method for assembling CRISPR constructs on demand. Assemblies are delivered ready-to-go for transformation and plasmid prep, followed by downstream genome editing applications.


VALIDATION DATE:  September 2015
PROTOCOLS EXECUTED:  Dilute Oligos; Anneal Oligos; Transform, Spread, Pick; Plasmid Maxi Prep; Linearize Vector; Assemble Constructs