Synthetic guide sequence to generate CRISPR-Cas9 entry strains in .

CRISPR/Cas9 genome editing has become an important and routine method in research to generate new mutants and endogenously tag genes. One complication of CRISPR experiments is that the efficiency of single-guide RNA sequences can vary dramatically. One solution to this problem is to create an intermediate entry strain using the efficient and well-characterised guide RNA sequence. This "d10 entry strain" can then be used to generate your knock-in of interest. However, the sequence is not always suitable when creating an entry strain. For example, if your gene of interest is closely linked to on LGII or if you want to use the as a co-CRISPR marker for the creation of the entry strain then you can not use the sequence. This publication reports a synthetic guide sequence, GCTATCAACTATCCATATCG, that is not present in the genome and can be used to create entry strains. This guide sequence is demonstrated to be relatively robust with a knock-in efficiency that varies from 1-11%. While this is lower than the efficiency observed with entry strains, it is still sufficient for most applications. This guide sequence can be added to the CRISPR toolkit and is particularly useful for generating entry strains where the standard guide sequence is not suitable.