This page gives a brief explanation of USER-cloning and USER-fusion.
Uracil-specific excision reagent (USER) is a enzyme mix containing:
- Uracil DNA glycosylase
- DNA glycosylase-lyase
Like Gibson Assembly, this assembly method requires short regions of identity between the sequences to be assembled so that adjacent sequences ‘overlap’ each other. It also requires the presence of uracil residues at the 3′ end of the overlap regions. The USER enzyme mix removes the uracil base and then breaks the backbone at the abasic site creating a 3′ overhang, which allows the adjacent fragments to self-anneal.
To assemble by USER cloning, a recipient vector must be adapted. This is done by cloning in a cassette consisting of a restriction enzyme recognition site (PacI in the diagram below) and two nicking-enzyme recognition sites (Nt.BbvCl in the diagram below). Two different nucleotides flank the restriction enzyme recognition site to allow directional cloning. The vector containing the USER cassette is then treated with a mixture of the restriction and nicking enzymes to create eight base pair 3′ overhangs.
The sequence to be inserted is amplified by PCR with oligonucleotide primers that contain 5′ eight base pair extensions with identity to the eight base pair overhangs in the receiving vector, except for the last thymine, which is replaced with a uracil. When the amplicon is treated with the USER enzyme mix, the uracil DNA glycosylase catalyses the excision of the uracil base causing the formation of an abasic site. The DNA glycosylase-lyase then breaks the phosphodiester backbone, releasing the base-free deoxyribose. This induces the formation of an eight base pair overhang, complementary to the vector (see diagram below).
IMPORTANT NOTE: Normal proof-reading polymerases will stall when they encounter a uracil residue in the template. To amplify fragments primed by oligonucleotides containing uracil residues, you must use an enzyme that is engineered to read through uracil bases such as PfuTurbo Cx , Phusion U or Kapa HiFi Uracil + (please note that we have not tried the latter product at TSL).
USER fusion is used to assemble multiple overlapping fragments. Overlapping double-stranded molecules are created by PCR amplification of each fragment, primed by custom oligonucleotides that include 5′ extensions with identity to the adjacent fragment. The overlap regions should be 6-17 bp long, start with and A and end with a T (see diagram below). As for single-insert assembly, the 3′ T is replaced by a U in the oligonucleotide used to amplify the insert. When the amplicon is treated with theUSER enzyme a 3′ overhang with identity to the adjacent fragment is created, allowing figments to self-anneal.
USER fusion allows scarless assembly of multiple fragments, however 13 bps of the USER cassette will still present between the vector backbone and the inserted fragments.
The self-annealed assemblies are transformed into competent E.coli where the single-strand nicks are repaired.