Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans

Abstract Highly efficient generation of deletions, substitutions, and small insertions (up to ∼150 bp) into the C. elegans genome by CRISPR/Cas9 has been facilitated by use of single-stranded oligonucleotide donors as repair templates. However, efficient insertion of larger sequences such as fluorescent markers and other functional proteins remains inefficient due to lack of standardized methods for generating repair templates and labor intensive or cost prohibitive synthesis. We have optimized the simple and efficient generation of long single-stranded DNA for use as donors in CRISPR/Cas9 using a standard PCR followed by an enzymatic digest by lambda exonuclease. Comparison of long single-stranded DNA donors to previously described methods using double-stranded DNA yields orders of magnitude increased efficiency for single-stranded DNA donors. This efficiency can be leveraged to simultaneously generate multiple large insertions as well as successful edits without use of selection or co-conversion (coCRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized and labor-minimal protocol.