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Homology Arm Calculator

Extract the left and right homology arms from the sequence around an insertion or edit point, with their GC content and approximate Tm, and assemble the donor with an insert.

Formula

left arm = the n bases ending at the edit point\text{left arm = the } n \text{ bases ending at the edit point}
right arm = the n bases starting after it\text{right arm = the } n \text{ bases starting after it}
donor=left arm+insert+right arm\text{donor} = \text{left arm} + \text{insert} + \text{right arm}
edit point\text{edit point}
the base after which the new sequence goes
nn
arm length in bases

How it works

Homology-directed repair and overlap assembly both use stretches of sequence that match the target on either side of the change. The left arm is the sequence immediately before the edit point, and the right arm the sequence immediately after it. Placed either side of an insert, they direct it to the right position.

Arm length depends on the method. Overlap-based assembly uses overlaps of a few tens of bases, single-stranded donors use arms of tens of bases, and plasmid donors for genome editing commonly use much longer ones. The composition of each arm is shown because arms with very low or very high GC content can be harder to assemble or synthesise.

Worked example

A 115 nt illustrative sequence with an edit after base 60 and 40-base arms.

  1. Left arm = bases 21 to 60.
  2. Right arm = bases 61 to 100.

Two 40-nt arms, with 50% and 53% GC, flanking the edit point.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The supplied sequence is the real sequence of the target, including any polymorphisms in your cells.
  • Arms are taken on the strand entered and read 5′ → 3′.

Common mistakes

  • Forgetting to change the PAM or the guide's target in the donor, which lets the nuclease cut the repaired allele again.
  • Taking arms from a reference genome when the cells differ from it in that region.
  • Counting from zero, so the arms are shifted by one base.

Related tools

Gibson Assembly CalculatorFragment amounts and volumes for an overlap assemblyOpen calculatorCas9 Target Finder and PAM FinderFind PAM sites and protospacers for SpCas9, SpCas9-NG or SaCas9Open calculatorOligo and Primer AnalyzerLength, GC, Tm, weight and a design-guideline check for a primerOpen calculatorPlasmid Size CalculatorSize of a recombinant plasmid after cloningOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.