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. 2019 Feb 21;73(4):727-737.e3.
doi: 10.1016/j.molcel.2018.12.015. Epub 2019 Jan 29.

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

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Free PMC article

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

Addison V Wright et al. Mol Cell. .
Free PMC article

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 mini-integrase 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.

Keywords: CRISPR; integrase; protein-DNA recognition; spacer acquisition.

Conflict of interest statement

DECLARATION OF INTERESTS

UC Regents has filed patents related to this work on which D.B., J.F.B., L.B.H., and J.A.D are inventors. L.B.H. is a co-founder of Mammoth Biosciences. J.F.B. is a co-founder of Metagenomi. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribous Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D is a member of the board of directors at Johnson & Johnson, and has sponsored research projects by Pfizer, Inc. and Biogen.

Figures

Figure 1.
Figure 1.. Type V-C and V-D CRISPR Systems Have Short Repeats and Spacers and Lack cas2 Genes
(A) CRISPR-Cas loci of the effector proteins of the type V-C system from mouse cecum and V-D system from beetle gut with lengths of genes and repeats and spacers. See Table S1 for protein sequences. (B) Distribution of mean spacer lengths of type V-C/D systems. (C) Comparison of mean spacer length for V-C/D vs. other CRISPR-Cas systems. The blue density plot depicts the distribution of mean spacer length in CRISPR-Cas systems, excluding V-C/D systems. The mean spacer lengths of published V-C and V-D systems are noted in red and orange dots, respectively, with arbitrary y-values to set them apart.
Figure 2.
Figure 2.. Type V-C Cas1 Integrates DNA Fragments of Expected Length In Vitro
(A) Schematic of in vitro integration of protospacer into a target supercoiled pCRISPR plasmid. Nucleophilic attack by the protospacer yields two different open-circle products: the half-site and full-site integration products. (B) Integration assay comparing type I-E, V-C, and V-D systems. A 33-nt protospacer is used for the type I-E system and an 18-nt protospacer for the type V-C and V-D systems. The open-circle integration products (OC), supercoiled target plasmid (SC), and topoisomers (Topo) are indicated. (C) Integration assay with type V-D Cas1, radiolabeled protospacer, and plasmid target. Integration product and free protospacer are indicated and schematized. (D) Integration assay with variable length fluorescent protospacers from 15-bp to 25-bp long. Star indicates 6-carboxyfluorescein label. (E) Quantification of (D) demonstrating the effect of protospacer length on type V-C Cas1 integration. The fraction integrated is calculated as the fraction of the fluorescent protospacer that has been integrated into the target plasmid. Experiments were carried out in triplicate; the bars represent mean values, with error bars depicting standard deviations. See also Figure S1. See Table S2 for nucleotide sequences.
Figure 3.
Figure 3.. Integration of Protospacers Occurs at Many Off-Target Sites
(A) Schematic of library preparation protocol for high-throughput sequencing. Integration products are fragmented, end-repaired, A-tailed, and ligated with Y-adapters. A protospacer-specific extension is carried out before amplification with Illumina primers. (B and C) Integration sites along pCRISPR. Results are separated based on the orientation of the protospacer that is integrated: the plots show integration by the (+) strand of the protospacer (B) and the (-) strand of the protospacer (C). See also Figures S2 and S3. (D and E) Magnified view of integration in the CRISPR arrays by the (+) strand of the protospacer (D) and the (−) strand of the protospacer (E). See Table S2 for nucleotide sequences.
Figure 4.
Figure 4.. Detection of Full-Site Integration by Type V-C Cas1
(A) Schematic representation of the chloramphenicol resistance turn-on screen to detect full-site integration near the leader-repeat junction. The construct contains a CRISPR repeat and leader upstream of an out-of-frame chloramphenicol resistance gene (CmR + 2). Translation of the transcript generated by Pcat begins upstream of the repeat (arrow) and ends in the leader (stop sign). Full-site integration of an 18-nt protospacer restores the open reading frame for the CmR coding sequence. Transforming and plating on chloramphenicol plates allows for positive selection of clones that have the inserted spacer. Sanger sequencing is used to confirm full-site integration. (B) Visual representation of identified full-site integration events near the leader-repeat junction. The colored arrowheads designate the position of the integration sites, with the number written above the arrowhead representing the number of base pairs from the leader-repeat junction. The corresponding colored lines designate the sequence that is duplicated upon spacer insertion. The arrowhead height is scaled to the total integration events at that site. (C) Number of integration events with specified spacer orientation at each integration site. The number of non-integration events (as a result of deletions) is also indicated. The mean and standard deviation of three independent replicates are shown. See also Figure S4. See Table S2 for nucleotide sequences.
Figure 5.
Figure 5.. Type V-C Cas1 Forms a Multimeric Complex for Integration
(A) Chromatograms of size-exclusion runs of type V-C Cas1, Cas1 with protospacer, Cas1 bound to half-site substrate, and Cas1 bound to pseudo-full-site substrate. Dashed black line indicates elution volume of free Cas1 dimer. Dashed green line indicates elution volume of Cas1 complex bound to full-site substrate. (B) Molecular weight characterization of the apo Cas1 and Cas1 bound to pseudo-full-site substrate by size exclusion chromatography coupled with dual angle light scattering. The experimental Mp for each peak is indicated. See Table S2 for nucleotide sequences.
Figure 6.
Figure 6.. Detection of Type V-C Cas1 Tetramer by Native Mass Spectrometry
(A) NanoESI mass spectra of cross-linked samples of apo Cas1. Measured molecular masses and corresponding cartoons of each species are listed on the top right corner. Ions of different species are labeled with different colors. (B) NanoESI mass spectra of cross-linked samples of Cas1 after complexing with pseudo-full-site substrate. See also Figure S6. (C) Model for type V-C mini-integrase compared to canonical Cas1-Cas2 integrase. See Table S2 for nucleotide sequences.

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