Red/ET & CRISPR/Cas
Add the advantages of recombineering to the power of CRISPR/Cas9 to extend your genome studies
CRISPR/Cas9 permits rapid knock-outs or site directed mutagenesis but is not well suited to larger and more complicated genome engineering exercises. These can be accomplished with Red/ET Recombination (recombineering) to generate targeting constructs or BAC transgenes that compliment CRISPR/Cas9 applications.
For example, instead of using CRISPR/Cas9 in sequential steps to introduce multiple mutations in a gene, each of which may need to be introduced into both endogenous alleles and then characterised in situ, it is less work with a more flexible outcome to introduce the mutations into a BAC transgene, which is introduced over a CRISPR/Cas9 knock-out.
In many cases, it is easier to do the precise work in E.coli using recombineering:
- well characterized BAC libraries exist for human, mouse and most model organism; annotated BAC clones available from different suppliers;
- BACs normally carry an entire genomic locus of a gene of interest including all necessary regulatory elements;
- Red/ET recombination offers fast and reliable way to modify BACs and to prepare an “allele” harbouring all interesting modifications of the gene of interest, such as humanization, multiple mutations, insertion of several SNPs, etc. → “designer BACs”.
Furthermore, BAC transgenesis can best be achieved by transposition:
- transposase mediated BAC transgenesis ensures full length, single copy insertions of BACs and the exact genomic insertion site can be readily identified e.g. by Splinkerette PCR;
- multi-use of BACs: modified BAC clones can be reused for insertion in other cell lines saving time and money in further analyses.