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

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, insertion of larger sequences such as fluorescent markers and other functional domains remains challenging due to uncertainty of optimal performance between single-stranded or double-stranded repair templates and labor-intensive as well as inefficient protocols for their preparations. Here we simplify the generation of long single-stranded DNA (ssDNA) as donors in CRISPR/Cas9. High yields of ssDNA can be rapidly generated using a standard PCR followed by a single enzymatic digest with lambda exonuclease. Comparison of long ssDNA donors obtained using this method to double-stranded DNA (dsDNA) demonstrates orders of magnitude increased insertion frequency for ssDNA donors. This can be leveraged to simultaneously generate multiple large insertions as well as successful edits without the use of selection or co-conversion (co-CRISPR) 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.