Optimizing recombineering with ssDNA

With ssDNA recombination it is possible to create single or multiple clustered point mutations (which can create a functional knockout of a gene), small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Multiple genes can be altered by using multiple oligos in the same reaction. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. A crucial discovery came in 2003 when Nina Costantino and Don Court found that avoidance or inactivation of the methyl-directed mismatch repair (MMR) system is key to optimization. In the absence of the MMR system, recombinant cells can be found in over 50% of unselected colonies by a PCR-based screen. The drawback of recombineering in cells mutant for the MMR system, however, is that these cells are mutagenic, allowing additional unwanted genetic changes elsewhere in the genome. The MMR system can be bypassed if the oligo, when paired to the target, creates a C/C mismatch either at or within 6 bases of the desired change. Altering 4-5 bases in a row or altering the wobble position of 4-5 adjacent codons in addition to the desired change will also avoid the MMR system and result in high recombination levels. The latter technique is especially powerful as changes in the wobble position do not alter the protein sequence, thus the technique can be used to mutate essential genes.

In summary, to find optimal recombination levels with ssDNA, an oligo should: 1) be from 40-70 bases in length with the desired change(s) near the middle of the oligo, 2) correspond to the lagging-strand of DNA synthesis, and 3) be designed to avoid the MMR system.