Nested CRISPR for cloning-free fluorescent tags
A better way to make endogenous reporters in C. elegans
CRISPR systems for gene editing have revolutionized biological research, but the method still has limitations. While it is usually straightforward to delete parts of the genome using CRISPR, large insertions can be a challenge. This has been the case even for the nematode Caenorhabditis elegans, one of the most established model organisms. But now, work published in GENETICS by Vicencio, Martínez-Fernández, Serrat, and Cerón has produced a more effective way to use CRISPR to insert longer stretches of DNA into the nematodes’ genomes.
A method for adding long DNA fragments is essential because many genes of interest, including important fluorescent reporter genes, are too long to be effectively inserted using existing methods. In fact, the team embarked on the work after attempts to insert a gene into the nematodes using another CRISPR-based technique repeatedly failed—a problem also reported in at least one other publication. In contrast, they found that their method, called Nested CRISPR, could efficiently add segments of DNA up to 792 base pairs long. They also achieved insertions of 927 base pairs, although the efficiency was lower.
In their method, the gene is inserted in two CRISPR-based steps. First, a short fragment with nucleotides from each end of the gene is inserted into the target site. Next, this fragment is replaced by the full-length insertion via homology-directed repair.
Their results mean that when insertions of a few hundred base pairs are needed, Nested CRISPR is a viable alternative to current methods involving extrachromosomal or randomly inserted DNA. The Nested CRISPR technique may even be broadly applicable to other organisms, particularly through the authors’ one-shot approach to achieve the two editing steps in a single injection.
It’s not completely clear why this group and others have had difficulty reproducing the level of efficiency reported for an existing CRISPR-based method for inserting DNA segments of this length into C. elegans. Slight differences in reagents among labs may be partially to blame for the lack of reproducibility of some laboratory methods, including those used for genome editing, but the authors of this study believe that won’t be an issue in the case of Nested CRISPR because all the reagents are commercially available and affordable. The availability of these premade reagents may also make it easier for researchers with less experience in gene editing (or molecular cloning in general) to perform the technique, allowing them to pursue projects that they otherwise may have avoided. The group has called for the C. elegans community to come together to evaluate the utility of methods such as theirs for inserting long stretches of DNA—which may become even more important as the field continues to hurtle forward.
Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
Jeremy Vicencio, Carmen Martínez-Fernández, Xènia Serrat, Julián Cerón
GENETICS 2019 211(4): 1143-1154; https://doi.org/10.1534/genetics.119.301965