RNA-triggered cell killing with CRISPR–Cas12a2 | Nature

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Subjects

- Biological techniques

- DNA damage and repair

Abstract

Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture1,2,3. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection4,5,6,7; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive8,9,10,11,12,13,14. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding15,16, and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging this approach, we selectively eliminate cells that harbour human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile.

Main

Selectively eliminating cells, tissues and organisms on the basis of their genetic or physiological identity has been a mainstay of the life sciences, whether for precisely removing diseased cells, shaping cellular communities, or eradicating contaminants or pathogens1,2,3. Common molecular and cell-based interventions, such as small-molecule inhibitors, toxins, antibodies, lytic viruses or programmed immune cells, eliminate cells through specific proteins or survival pathways17,18,19,20; however, these methods cannot be tailored to arbitrary genetic or transcriptional states as well as difficult-to-drug scenarios such as mutations in non-coding sequences or complex aetiologies. A cell-killing approach triggered directly by the specific recognition of prescribed DNA or RNA sequences could greatly broaden the range of targetable conditions, creating new means to counterselect against specific cells in a variety of situations and applications.

CRISPR nucleases—RNA-guided effector proteins of CRISPR–Cas immune systems in bacteria and archaea21—have various biochemical activities triggered by recognizing targeted DNA or RNA; such activities could be harnessed to selectively eliminate cells on the basis of their genetic or transcriptional state22,23,24,25. In bacteria, CRISPR nucleases potently eliminate cells containing a target sequence via different mechanisms; for example, Cas9 and Cas12a introduce targeted double-stranded DNA (dsDNA) breaks that are poorly repaired4,5, or Cas13a collaterally cleaves RNA driving cell dormancy6,7. However, these activities are largely ineffective for programmed cell elimination in eukaryotic cells. Instead of resulting in cell death, dsDNA breaks by Cas9 or Cas12a are efficiently repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ)26,27,28. Only when targeted cleavage occurs throughout the genome, such as by targeting highly repetitive elements, can these nucleases induce cell death12,13,14. Separately, activating Cas13 in eukaryotic cells usually leads to specific degradation of the target transcript10. When indiscriminate RNA degradation has been observed, activated Cas13 fails to drive robust cell dormancy or death8,9,10,11. Thus, CRISPR-based approaches that broadly enable selective cell elimination in bacteria remain elusive in eukaryotes.

We recently reported a clade of RNA-guided CRISPR nucleases called Cas12a2 that, upon specifically base pairing with complementary RNA, unleashes indiscriminate dsDNase activity that drives an SOS DNA damage response and cell dormancy in bacteria15,16. However, the effect of Cas12a2’s RNA-triggered indiscriminate dsDNase activity remained unexplored in eukaryotes. We thus asked how Cas12a2 affects eukaryotic cells when unleashed by specific recognition of a transcript and whether these nucleases can be used for RNA-triggered programmable cell elimination.

Cas12a2 eliminates yeast cells

To explore the effect of triggering Cas12a2 nucleases in eukaryotic cells, we used two closely related variants of Cas12a2: one from Sulfuricurvum sp. PC08-66 (SuCas12a2), and another derived from a metagenomic sample (GeCas12a2). The enzymes share 89% identity (Extended Data Fig. 1) and have similar biochemical properties, including recognition of RNA targets upstream of an adenine-rich protospacer-flanking sequence (PFS) and subsequent collateral cleavage of RNA, single-stranded DNA and dsDNA (Fig. 1a and Supplementary Fig.  2, Extended Data Figs. 1 and 2, and Supplementary Table 1), lending themselves to the same strategy for guide RNA (gRNA) design (Extended Data Fig. 3).

Fig. 1: RNA-triggered Cas12a2 eliminates yeast and