A Functional Mini-Integrase in a Two-Protein Type V-C CRISPR System

Abstract

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a miniintegrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17-to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

“…We next set out to determine whether the Cas6-RT-Cas1-Cas2 complex can catalyze full-site CRISPR spacer integration. We devised an assay using chloramphenicol resistance as a selection marker for plasmids with protospacer insertion events near the target region 52,53 . The reporter construct was designed with 163 nt of the leader sequence and the CRISPR repeat sequence immediately upstream of a chloramphenicol resistance gene with a missing ribosomal binding site (RBS) sequence and start codon ( Fig.…”

CRISPR-Cas Systems Reduced to a Minimum

“…Deep sequencing of integration events revealed a surprisingly high frequency of off-target spacer integration into non-CRISPR loci, which could indicate the requirement of a yet unknown host factor or sequence element for specificity in the natural host. Wright et al (2019) further back their findings by employing plasmids reporting successful integration of new spacers (Díez-Villaseñ or et al, 2013) and made the interesting observation that Cas1 intrinsically determines spacer orientation and defines the location of integration depending on the protospacer sequence. Size-exclusion chromatography and mass spectrometry revealed the formation of a tetrameric Cas1 assembly in association with mimics of spacer integration intermediates.…”

“…Interestingly, the spacer length found in both systems is considerably smaller (18-20 bp) than in all other CRISPR-Cas systems harboring cas2. Wright et al (2019) present compelling in vitro evidence that Cas1 proteins from both systems support spacer integration using purified Cas1 and plasmid DNA containing the cognate type V CRISPR array. The type V-C Cas1 (Cas1c) displayed intrinsic preference for prespacers of just 18 bp in length, which is almost half the size of most spacers in CRISPR systems encoding Cas2.…”

“…Recently in Molecular Cell, Wright et al (2019) report the exception to that rule. They identify two type V CRISPR-Cas systems in metagenomes from a mouse and beetle gut that both lack the cas2 gene.…”

“…In two recent studies in Molecular Cell, Wright et al (2019) report complete spacer integration by a Cas1 miniintegrase and Edraki et al (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.…”