Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in .

CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans. This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (<= 200 bp) are used to insert the 5 ' and 3 ' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.