How do I simulate NEBuilder Hifi DNA Assembly® with a single stranded insert?
NEBuilder Hifi DNA Assembly® is a registered trademark of New England Biolabs Inc. For information about NEBuilder Hifi DNA Assembly® see this link.
In this lesson we will simulate NEBuilder Hi Fi DNA assembly to "clone" an single stranded oligonucleotide, comprising a CRISPR guide with vector-specific flanking overlaps, into an RNA expression vector.
This lesson follows the guidelines published by NEB, see - Hsieh, Application note.
Open or Import a Vector Sequence
In this example, File → Import → AddGene Plasmids is used to import AddGene sequence number 42230, a chimeric guide RNA expression plasmid with a human codon-optimized SpCas9.
Import or open a sequence file for the plasmid vector that will be used for NEBuilder HiFi DNA Assembly®.
Display the Sites for Linearization of the Vector
If required, click Enzymes → Choose Enzymes to display the enzyme/s sites that will be used for linearization of the vector.
In this example BbsI sites will be used to linearize the vector and allow placement of a CRISPR guide between the U6 promoter and CRISPR scaffold.
Identify Vector Regions to Use for Overlap and Assembly
Switch to Sequence view and identify the insertion point. In this example we have added three features, "Left", "Region to Replace" and "Right". See Create a Simple Feature.
The "Left" and "Right" features are each 25 bp long and represent the overlap that will be added to the CRISPR guide sequence to facilitate Hifi Assembly.
The "Region to Replace" feature marks the vector region that will be replaced by the incoming guide insert.
Create the Insert Sequence
Create or open a sequence that represents your CRISPR guide sequence. In this example we have added and segmented a feature to show the spacer and PAM (NGG) components of the guide sequence.
Switch to the vector sequence and select the "Left" feature and go menu Edit → Copy.
Switch to the insert sequence. Click to place the cursor to the left of position one then click menu Edit → Paste.
Copy the "Right" feature from the vector sequence.
Switch to the insert sequence.
Select the region representing the "NGG" PAM sequence.
Click Edit → Paste to replace the PAM sequence with the "Right" feature.
Make sure the PAM site is replaced by the "Right" sequence!
The insert sequence will now comprise the guide sequence (without PAM), flanked by the left and right regions that will be used for NEB HiFi assembly.
Click menu Actions → Convert to Single Stranded.
Save the new Single stranded sequence file. This sequence represents the oligonucleotide to order to cloning.
Start the NEBuilder HiFi DNA Assembly® Tool
Set the Vector "Region for Replacement"
Switch to the vector sequence then click Actions → NEBuilder HiFi DNA Assembly® → Insert Fragment....
The NEBuilder HiFi DNA Assembly® tool opens showing the "Vector" tab. Click on the leftmost BbsI site, then shift-click on the rightmost BbsI site to select the BbsI fragment that will be replaced during NEB Hifi assembly. The cloning tool will automatically set the option to "Linearize with restriction enzymes".
Set the Insert Fragment
Switch to the "Fragment" tab.
Use the dropdown to load the single stranded DNA fragment as the fragment.
Click on the sequence and click menu Edit → Select All to specify the entire sequence as the insert fragment.
Select the option to "Use Directly as the Fragment".
SnapGene will identify that the selection has overlaps suitable for assembly with the vector. The panel will turn green indicating assembly can proceed.
Validate the Vector/Insert Fusion
Switch to the Product tab.
Switch to Sequence view and confirm that the guide insert is correctly placed in the vector.
Enter a name for the product file, then click Assemble to create the product sequence.
Click Assemble to create the new product file.
View the Product File
View the product file.
Click File → Save to save the new sequence to an appropriate location on your computer.
View the Product History
Switch to History view to see all steps simulated during the NEBuilder Hifi DNA Assembly® procedure.
Order the Oligonucleotide
Open the single stranded sequence that was created for use as the insert in the above procedure.
Go Edit → Select All and copy the full sequence (5'-3') for ordering from your preferred oligosynthesis service.
