UC Berkeley

In the ongoing arms race between prokaryotes and phage, the emergence of CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR-associated) adaptive immunity provided bacteria and archaea a unique basis of defense against genetic invaders. CRISPR systems record immunological memory of previous infection in the host CRISPR genomic locus, consisting of alternating short repeat sequences and foreign-derived spacers. During CRISPR adaptation, fragments of foreign genetic material (prespacers) are captured and processed into short sequences (protospacers) that are integrated into the CRISPR array. After transcription of the CRISPR array and maturation of CRISPR RNAs (crRNAs), CRISPR interference proteins will associate with individual crRNAs to carry out target identification and destruction. This work describes the origins and evolution of the CRISPR adaptation module and the diversity of enzymes and mechanisms used in spacer acquisition.

The core component of the CRISPR adaptation module is the CRISPR integrase, which in most systems, consists of the Cas1 and Cas2 proteins and performs the integration of new spacers. We identify a functional CRISPR mini-integrase comprising of only the type V-C Cas1 that may represent the ancestral CRISPR integrase prior to Cas2 adoption. We determine that the mini-integrase forms a tetramer for integration and has selectivity for short 17-19 bp substrates. Based on the evolutionary model, the mini-integrase may mark the emergence of a precise ruler mechanism dictating spacer length, a hallmark of CRISPR integrases.

In certain CRISPR systems, a reverse transcriptase (RT) is another key component of the adaptation module and enables spacer acquisition from RNA sources. Using cryo-electron microscopy, we uncover the structure of a naturally occurring fusion complex that combines an RT, a Cas1—Cas2 integrase, and Cas6 maturase and through structure-guided mutagenesis, we reveal crosstalk between the three active sites. Based on a unique RT conformation and other domain interactions, we suggest structural rearrangements that may coordinate sequential enzymatic activities enabling spacer acquisition from RNA and DNA substrates.

A third class of enzymes involved in CRISPR adaptation are the enzymes that aid in the processing of prespacers into viable integration substrates. In many systems, this involves the removal of the protospacer adjacent motif (PAM), which serves to distinguish between self and non-self. Using a natural DEDDh exonuclease fusion to a Cas1—Cas2 integrase, we elucidate the stepwise coordination of prespacer processing and integration and reveal a gate-keeping ruler mechanism providing exquisite precision in PAM removal. These results underscore the diverse structures and mechanisms employed in the generation of CRISPR immunological memory.