Tracing the origins of CRISPR–Cas

Understanding the evolutionary origins of CRISPR–Cas and other RNA-guided immune systems could reveal new mechanisms for programmable genetic manipulation. Writing in Science, Yoon and colleagues describe viral interference programmable repeat (VIPR) systems, a family of RNA-guided DNA-targeting systems that may represent evolutionary precursors to class 1 CRISPR–Cas.

Using structure-based searches of the AlphaFold database, the authors identified VIPR systems predominantly in bacteriophages. Small-RNA sequencing and protein pull-down experiments showed that these systems comprise a small Vipr protein bound to a VIPR RNA that contains tandem repeats in which conserved GGY motifs alternate with variable NN dinucleotides (in which G is guanine, Y is a pyrimidine and N is any nucleotide). Analysis using the Evo2 genomic language model suggested that the NN bases recognize DNA through a non-contiguous code in which pairs of matched bases are separated by an unpaired nucleotide. The authors validated this ‘one-nucleotide skip’ rule using in vitro DNA-binding assays and transcriptional repression experiments in Escherichia coli. Changing the NN sequences redirected VIPR towards new targets on either DNA strand, without an obvious requirement for the protospacer-adjacent motif used by many CRISPR–Cas systems.

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