NEW YORK (GenomeWeb) – Researchers from the National Institutes of Health and the Broad Institute have developed a system for precisely inserting DNA into a genome that uses a CRISPR-associated transposase (CAST).
As they wrote today in Science, the researchers, led by the Broad's Feng Zhang and the NIH's Eugene Koonin, characterized a CAST from the cyanobacterium Scytonema hofmanni. The system, which they called ShCAST, consists of Tn7-like transposase subunits and the type V-K CRISPR effector Cas12k.
"ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 bp downstream of the protospacer," the authors wrote. "ShCAST integrates DNA into unique sites in the E. coli genome with frequencies of up to 80 percent without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion."
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, the researchers noted. The team hypothesized that these limitations could be overcome using self-sufficient DNA insertion mechanisms, such as transposons.
Recent studies have reported an association between Tn7-like transposons and subtype I-F, subtype I-B, or subtype V-K CRISPR-Cas systems. "The association between Tn7-like transposons and CRISPR-Cas systems suggests that the transposons might have hijacked CRISPR effectors to generate R-loops in target sites and facilitate the spread of transposons via plasmids and phages," the authors added. "In the case of subtype V-K, the position of the CRISPR-Cas locus is frequently conserved in predicted transposons, suggesting that CRISPR-Cas is linked with transposition."
The researchers found that Tn7-like transposons could be directed to target sites via crRNA-guided targeting, and that Tn7 transposition could be reprogrammed to insert DNA into the genome of E. coli. Of the 560 spacers they analyzed in subtype V-K CRISPR-Cas systems, they only identified six protospacer matches, suggesting that V-K systems provide a biological advantage for the host transposons by directing integration into other mobile genetic elements, to enhance transposon mobility, and to minimize the damage to the host.
For experimental characterization, the investigators selected two Tn7- like transposons encoding subtype V-K CRISPR-Cas systems. The selected CAST loci were 20 kb to 25 kb in length and contained Tn7-like transposase genes at one end of the transposon with a CRISPR array and Cas12k on the other end. Through various experiments, the researchers found that the CAST from S. hofmanni could target a wide range of DNA sequences with minimal targeting rules.
To test whether ShCAST could be reprogrammed as a DNA insertion tool, they selected 48 targets in the E. coli genome and co-transformed the bacterium with two plasmids expressing single guide RNAs designed to target sites in the E. coli genome. Using PCR, they detected insertions at 29 out of the 48 sites (60.4 percent). They also performed ddPCR to quantitate insertion frequency after 16 hours and measured rates up to 80 percent.
They also assayed the specificity of the RNA-guided DNA transposition and found off-target reads scattered across the genome. Analysis of the top off-target sites revealed strong overlap between samples, suggesting that these events were independent of the guide sequence, the researchers said. Further, top off-target sites were located near highly expressed loci such as ribosomal genes, serine-tRNA ligase, and enolase, although insertion frequencies in these regions were all less than 1 percent of the on-target site.
"Together, these results indicate that ShCAST robustly and precisely inserts DNA into the target site," they added. "ShCAST mediates unidirectional insertions in a narrow window downstream of the target and inhibits repeated insertions into a single target site."
Further studies are needed to improve understanding of the function of each transposase subunit in the CAST complex, and future protein engineering of the transposase components could improve the targeting specificity of CAST systems, the team noted, concluding that the study provides a strategy for targeted insertion of DNA without engaging homologous recombination pathways, "with a particularly exciting potential for genome editing in eukaryotic cells."