Primer and CRISPR Design

Three tools design what you are about to order: PCR primers for a region you want to amplify, CRISPR guides against a target, and a codon rewrite for the organism you are expressing in. All three work on DNA, and all three open from the selection menu, so you pick the bases first and the tool arrives already pointed at them.

Drag out the region, then choose the tool from the menu. Plasmid Maps and the Sequence Viewer covers that menu and the rest of the viewer.

Designing Primers

Design Primers sits under Create. It opens Primer Design with your selection already in Target Region (region to amplify), as a start and an end you can still edit. Open it without a selection and it aims at the middle of the sequence, which is a starting point rather than an answer.

Four parameters shape the search:

Melting temperatures come from the nearest-neighbour thermodynamics you would expect, with a salt correction, so the numbers line up with what your ordering sheet says.

Design Primers runs the search and returns up to five pairs, the closest match first, each with a score, the product size, and the difference in melting temperature between the two primers. A pair whose primers disagree by more than 5 °C is rejected outright. Each primer shows its Tm, GC content, length, and its sequence written 5' to 3'.

Anything questionable about a primer is called out on the pair itself: a weak 3' clamp, a hairpin, a self-dimer, a repeat run, GC or Tm outside your bounds. The pair still appears, so you can decide whether the flaw matters for this reaction.

Copy forward and Copy reverse take one primer to the clipboard. Add as annotation writes both onto the map as Primer Binding Site features named for their direction and melting temperature, such as Forward Primer (60.1°C), which is how the pair you actually ordered stays visible next time you open the construct. Cmd/Ctrl + Z takes them back off.

When nothing comes back, the panel says which constraint was the binding one, so the next attempt is a considered change rather than a guess.

Designing CRISPR Guides

CRISPR Design sits under Analyze and opens CRISPR Guide RNA Design.

CRISPR System picks the enzyme, and with it the PAM and the guide length:

SpCas9 is selected for you. Target Region arrives from your selection and stays editable, and GC Content Range (%) starts at 30 to 70. Find Guide RNAs searches both strands inside that window and drops any protospacer carrying a run of more than four T's, since those terminate transcription of the guide.

The results are counted as high, medium, and low quality, and each guide shows its score, its strand, its sequence with the PAM, its position, and its GC content. Opening one breaks the score into what produced it: GC content, where the cut lands in your target, the seed region next to the PAM, the poly-T penalty, and self-complementarity.

Finding nothing usually means the window is too tight, and the empty state names the exact constraint to loosen. Add on a row, or Add as Annotation in the detail panel, writes the guide onto the map as a Feature named gRNA with its score.

Codon Optimization

Codon Optimize, also under Analyze, rewrites your selection for expression in another organism. Codon Optimization offers six hosts: E. coli, Yeast, Human, Mouse, CHO Cells, and Insect (Sf9). E. coli is chosen for you.

Before you run anything, the dialog box reads out the selection as it stands: its codon adaptation index for the host you picked, how many rare codons it contains, and how many codons there are in total. That alone answers whether optimizing is worth doing.

Use only most frequent codon per amino acid takes the single best codon every time. Left off, the rewrite keeps some of the natural variety, which avoids the long identical runs that make a gene hard to synthesize.

Optimize Sequence shows the result before it touches anything: the adaptation index before and after, the improvement between them, how many codons changed, the new GC content, how many rare codons survived, and the first fifty changes written out as old codon against new. Stop codons and anything it cannot read are left alone.

Apply Optimized Sequence writes the new bases over exactly the range you selected and leaves the rest of the sequence untouched, down to a trailing partial codon it puts back so the length never moves.