A novel approach for targeted genomic insertion of DNA fragments

Researchers from the National Institutes of Health and the Broad Institute report a novel system for precisely inserting DNA into a genome that uses a combination of a CRISPR-associated transposase (CAST) and a CRISPR effector protein (Cas12k) from the cyanobacterium Scytonema hofmanni.

Their system, termed "ShCAST" catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 bp downstream of the protospacer site with high efficiency. Inserting DNA fragments into the E. coli genome via ShCAST yielded insertion efficiencies of up to 80 percent, without the need for positive selection.

CRISPR-Cas nucleases have developed into powerful tools for genome manipulation. However, targeted insertion of DNA still remains a challenge as it requires the host cell repair machinery to achieve insertion via homologous recombination. Although it is possible to integrate new DNA in a genome following cleavage of a DNA strand by Cas9, this requires either homologous recombination or non-homologous end-joining, processes that are inefficient and vary greatly depending on cell type. Homologous recombination-based repair is also tied to active cell division, making it unsuitable for post-mitotic cells. 

Writing in Science, Strecker et al. now report a novel approach to targeted DNA insertion which circumvents the need for homologous recombination or NHEJ. Instead of relying on the host cell repair machinery for integration of exogenous DNA after CRISPR/Cas cleavage, they take advantage of a Tn7-like transposon combined with a subtype V-K CRISPR-Cas system from the cyanobacterium Scytonema hofmanni.

They found that Tn7-like transposons could be directed to target sites via crRNA-guided targeting, and were able to efficiently insert DNA into the genome of E. coli. Transforming bacteria with plasmids expressing 48  different single guide RNAs designed to target sites in the E. coli genome, they detected insertions at 29 out of the 48 sites by PCR. They also performed ddPCR to quantitate insertion frequency after 16 hours and measured rates of up to 80 percent.

On the downside, they also identified off-target insertions scattered across the genome. These off-target events were apparently independent of the guide sequence and were located near highly expressed loci such as ribosomal genes, serine-tRNA ligase, and enolase. Off-target insertions are a common problem with established CRISPR/Cas systems, although a number of high fidelity Cas9 and Cas12 mutants have been developed to alleviate these problems. Possibly, directed evolution approaches optimizing both the Tn7-like transposon and subtype V-K CRISPR-Cas protein may yield more specific variants in the future.

Clearly, further studies are needed to better understand the function of each transposase subunit in the CAST complex but these encouraging results indicate that ShCAST is capable of precisely inserting DNA into different target sites and may, in the future, also be used for targeted genomic insertions in other species.

Strecker et al. Science  06 Jun 2019: eaax9181 DOI: 10.1126/science.aax9181