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Broad, MIT Team Reports on Inducible CRISPR/Cas9 Approach

NEW YORK (GenomeWeb) – Researchers from the Broad Institute and the Massachusetts Institute of Technology this week reported on a new technique that enables inducible gene editing and transcription modulation via CRISPR/Cas9.

CRISPR — short for clustered, regularly interspaced, short palindromic repeats — involves the use of a nuclease known as Cas9 to induce double-strand DNA breaks (DSBs). These breaks are targeted to specific locations in the genome using a synthetic RNA, known as a guide RNA, that directs Cas9.

In a letter published in Nature Biotechnology, the Broad's Feng Zhang and colleagues described a method by which Cas9 can be split into two fragments that can be reassembled into its functional form using rapamycin-binding dimerization domains.

To do so, the team identified 11 potential sites where Cas9 could be split to create a series of fragment pairs — a C-terminal Cas9 fragment and an N-terminal Cas9 fragment — fused to the FK506-binding protein 12 (FKBP) and the FKBP rapamycin-binding domains of the mammalian target of rapamycin, respectively.

All 11 sets of split Cas9 were then tested by targeting the EMX1 locus in human embryonic kidney cells. "We detected insertion/deletion mutations mediated by all split-Cas9 sets in cells treated with rapamycin," the investigators wrote.

Moderate levels of indels were also detected in the absence of rapamycin, however — an effect found to be caused by the auto-assembly of Cas9 fragments in cells. To address this, Feng et al. sequestered the Cas9 fragments in the cytoplasm by replacing the two nuclear localization sequences (NLS) on one of the fragments with a single nuclear export sequence (NES).

In the presence of rapamycin, the NLS fragment dimerizes with the NES fragment to reconstitute a complete Cas9 protein, the investigators wrote, "which shifts the balance of nuclear trafficking toward nuclear import and allows DNA targeting."

In further testing, Feng's team found that the split Cas9 approach was able to induce "substantial indels at a targeted locus" without resulting in the off-targeting that frequently occurs with high doses of constitutively active Cas9.

They also demonstrated that the split-Cas9 architecture could be applied to catalytically inactive Cas9 to mediate inducible transcription activation, an effect that persisted even after the withdrawal of rapamycin. Given this, the scientists noted that their system could be useful for experiments where synchronized transcriptional activation is beneficial, such as cellular differentiation or development or modulation of genes that adversely affect the health or growth of the cell.

The split Cas9 approach may also prove useful for a variety of other applications, the researchers concluded.

"For example, split-Cas9 systems may enable genetic strategies for restricting Cas9 activity to intersections of cell populations by putting each fragment under a different tissue-specific promoter," they wrote in Nature Biotechnology. "Additionally, different chemically inducible dimerization domains, such as abscisic acid or gibberellin-sensing domains, may also be employed to generate an array of inducible Cas9 molecules, fused to different modulatory domains, to construct synthetic transcriptional networks."