Other editing & transgenesis
Pairs of transcription-activator-like effector nucleases: one TALE repeat per base of target, fused to half of FokI. Targeting is essentially unrestricted because no motif is required, which is their advantage over any guide-RNA tool; the arrays are repetitive and slow to build, which is their cost.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
TALENs
Pairs of transcription-activator-like effector nucleases: one TALE repeat per base of target, fused to half of FokI. Targeting is essentially unrestricted because no motif is required, which is their advantage over any guide-RNA tool; the arrays are repetitive and slow to build, which is their cost.
How it differs from the standard method
- Spacer between half-sites of roughly 12–21 bp, depending on the scaffold used.
- 5-methylcytosine blocks the repeat that reads an unmethylated C, so methylated targets need repeat variants chosen for it.
- Sequence the assembled arrays: tandem repeats lose and duplicate units during cloning.
1Why these are still in use
CRISPR did not make the older ways of modifying a genome obsolete; it made them specialised. Each of the approaches here does something a Cas9 nuclease cannot.
Dimeric nucleases
Two monomers, each a programmed DNA-binding domain fused to half of the FokI nuclease. Neither cuts alone; the pair cuts the spacer between them.
Site-specific recombination
Cre swaps DNA between two loxP sites. Their orientation decides whether the DNA between them is excised or inverted.
Transposition
A transposase excises the cassette between two inverted repeats and inserts it at its own short target motif somewhere in the genome.
2Programmable nucleases that come in pairs
A zinc finger reads three bases of DNA; a TALE repeat reads one. String four to six fingers, or seventeen repeats, together and you have a protein that binds one chosen sequence.
3Cre-lox: deletion where and when you choose
Cre recombinase recognises a 34 bp loxP site and recombines two of them. Nothing is cut, nothing is repaired, and no cellular repair pathway is involved, which is why it works in any cell, dividing or not.
4Transposons: room, at the cost of control
A transposase recognises the inverted repeats flanking a cassette, excises it and inserts it at a short motif elsewhere: TTAA for piggyBac, TA for Sleeping Beauty. Supplied in trans, as mRNA or from a plasmid, with the cassette on an ordinary donor plasmid, this gives stable integration with no viral packaging limit, no helper virus and very little work.
5How they fail
- A dimeric nuclease pair with the wrong spacer: both monomers bind, no active dimer forms, nothing is cut.
- Wild-type FokI or a large monomer excess: one monomer pairs with itself and cuts single sites.
- A TALEN array that lost a repeat during cloning, so it reads a sequence one base out.
- loxP sites built facing each other, giving a reversible inversion instead of a deletion.
- A Cre driver whose published expression pattern is broader than advertised, so the knockout is not conditional; or germ-line recombination quietly turning the colony into a conventional knockout line.
- An inducible CreER never given tamoxifen, which looks exactly like a failed allele.
- A transposon cassette judged at two weeks, before position-dependent silencing has had time to appear, or a pool used as if it were a clone.
Key terms
Practise these as flashcards →The nuclease domain used by TALENs and ZFNs. Only active as a dimer, so cutting requires two monomers bound the right distance apart.
The base pairs between the two half-sites. About 5–7 bp for zinc-finger nucleases and 12–21 bp for TALENs; outside that range no active dimer forms.
FokI mutated so that each monomer can only pair with its partner, not with a copy of itself. The standard way to cut off-target cleavage.
A ~34-amino-acid unit that reads a single base. One repeat per base, so a target needs no motif, but the arrays are highly repetitive to build.
A 34 bp sequence Cre recognises: two inverted 13 bp arms around an 8 bp asymmetric spacer, which is what gives the site its direction.
An animal line expressing Cre from a chosen promoter. Its expression pattern, not the floxed allele, decides where and when deletion happens.
An allele with loxP sites in introns around a critical exon: normal until Cre arrives, null afterwards.
Transposition falling as transposase rises past an optimum. Strong for Sleeping Beauty, so the enzyme-to-donor ratio has to be titrated.
Expression of an integrated cassette depending on where it landed, which is why clones from one transfection differ and drift apart over weeks.
When to use it
- The target has no usable PAM within reach, so no Cas9-based tool, including base and prime editors, can be pointed at it.
- You need a gene removed in one tissue, or at one age, in a living animal: a conditional Cre-lox allele is still the standard way to do it.
- You need a large cassette, more than a virus will carry, integrated into a cell line quickly and without viral work.
- You want a built-in two-site requirement for specificity, as in some therapeutic programmes where a single guide-directed cut is considered too permissive.
Compared with alternatives
Use Cas9 nuclease editing whenever a PAM is available: a guide RNA is an oligonucleotide order rather than a pair of protein arrays, and efficiency is usually at least as good.
Prefer a base or prime editor when you want a defined small change without a double-strand break, but note they still need a PAM, so a site out of Cas9's reach is out of theirs too.
For asking which of many genes matters, a pooled screen does in one experiment what building hundreds of nuclease pairs never could.
Transposon delivery is one route to a stable line; compare it with random plasmid integration and with viral transduction on cargo size, copy number and effort.
A Cre-lox colony lives or dies by its genotyping: the three-primer PCR that tells wild-type from floxed from deleted alleles belongs there.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Pairs of transcription-activator-like effector nucleases: one TALE repeat per base of target, fused to half of FokI. Targeting is essentially unrestricted because no motif is required, which is their advantage over any guide-RNA tool; the arrays are repetitive and slow to build, which is their cost.
- Spacer between half-sites of roughly 12–21 bp, depending on the scaffold used.
- 5-methylcytosine blocks the repeat that reads an unmethylated C, so methylated targets need repeat variants chosen for it.
- Sequence the assembled arrays: tandem repeats lose and duplicate units during cloning.