Updating the CRISPR Catalogue

Abstract

A new study provides the missing pieces toward understanding how bacteria upgrade their immunity.

Bacterial CRISPR-Cas adaptive immunity enables a rapid response to obliterate phage infection.¹ To keep pace with rapidly evolving phages, host cells upgrade their CRISPR array with information about their pathogen. Short snips of DNA are taken from the phage genome and integrated as “spacers” into the host CRISPR (Fig. 1).² Spacers serve as templates for CRISPR (cr)RNAs that guide Cas effectors to bind and destroy the phage genome.¹ Thus, adaptation to infection through spacer uptake immunizes a cell and its progeny against future infection.

FIG. 1.

How bacteria upgrade their immunity. The overall reaction is shown in the lower right corner, where the CRISPR array containing repeating sequences (gray diamonds) separate spacers (colored squares). The Cas1–Cas2 complex begins the upgrade by selecting single-stranded DNA degradation products containing a PAM sequence. Cas1–Cas2 facilitates pairing of the complementary strand to create a partially double-stranded substrate. DnaQ enzymes trim the 3′ ends of the substrates, but Cas1–Cas2 protects the PAM end of the DNA, creating an asymmetrical product. Partial trimming of the PAM delays integration of the PAM end, which only occurs after the non-PAM end has been integrated, ensuring the orientation of integration. Following resolution of the fully integrated spacer through DNA repair, the CRISPR array has been upgraded with a new spacer (blue square).

Over the past 10 years, adaptation has been studied extensively, with the type I-E system from Escherichia coli serving as an important model system. Early genetic studies revealed that spacer acquisition requires sequence elements within the CRISPR array as well as the cas1 and cas2 genes.³ Structural and biochemical studies revealed that Cas1 and Cas2 proteins form a complex that measures DNA substrate length and catalyzes a two-step integration reaction at the first repeating sequence within the CRISPR array.^4–6

Still, many steps that must occur prior to integration remained poorly defined. Prespacer substrates are thought to be generated through various DNA degradation pathways,^7,8 but how are they selected by Cas1–Cas2 and processed for integration? Moreover, how does Cas1–Cas2 ensure that the spacers formed during integration will encode functional crRNAs?

In a new study published in Nature,⁹ Chirlmin Joo and colleagues at the Delft University of Technology in The Netherlands answer numerous long-standing questions on how prespacers are generated, selected, processed, and integrated to form functional spacers.

First, the authors designed an elegant single molecule assay to monitor how Cas1–Cas2 can generate prespacers from DNA degradation products that are readily available in a bacterial cell. Cas1–Cas2 substrates must be at least partially double stranded. However, degradation products of RecBCD and CRISPR-mediated interference, which have been shown to serve as prespacers, are single stranded.^7,8 So, how does Cas1–Cas2 use these ssDNA products to produce double-stranded prespacers?

The authors discovered that while Cas1–Cas2 can stably bind to dsDNA substrates, it does not remain bound to ssDNA. However, in the presence of a complementary strand of ssDNA, Cas1–Cas2 can pair the two strands, creating a DNA duplex (Fig. 1). Prespacers must also contain a protospacer adjacent motif (PAM), which is required for Cas effectors to locate and bind their target during immune response.² Importantly, Cas1–Cas2 preferentially selects and anneals ssDNA strands containing PAM sequences, generating a suitable substrate (Fig. 1).

Next, the authors tackled questions about how Cas1–Cas2 ensures that the spacer will encode a functional crRNA. Following substrate selection, the PAM needs to be precisely removed from the prespacer prior to integration.² In most type I systems, an additional adaptation protein, Cas4, recognizes and removes the PAM, ensuring spacer fidelity.^10–14 However, type I-E systems lack Cas4. So, how is the PAM removed?

A Quick Trim

The authors hypothesized a role for cellular exonucleases in cutting the prespacer down to the right size. After testing various 3′-5′ exonucleases, they found that DnaQ exonucleases can trim prespacers (Fig. 1). Surprisingly, the authors observed asymmetric trimming, depending on the sequence present at each end of the prespacer. A strand lacking a PAM was trimmed all the way down to the length needed for integration. Meanwhile, the strand containing a PAM was only partially trimmed. What causes this asymmetry? The authors inspected the structure of Cas1 and deduced that the C-terminal tail of the protein may protect the PAM region. Indeed, mutations in this tail region removes Cas1 protection of PAM and results in symmetrically trimmed prespacers.

Formation of a functional spacer also requires integration of the prespacer in the correct orientation to act as a proper template for the crRNA.² However, Cas1–Cas2 integrates symmetrically trimmed prespacers in a random orientation. So, how is spacer orientation defined? The authors speculated that asymmetric prespacer trimming may delay integration of the PAM-containing strand (Fig. 1). This would enable integration of the non-PAM strand first at one end of the repeat, followed by integration of the PAM strand on the opposite end. Indeed, the authors observed that asymmetrical trimming ensures that the PAM strand is only integrated at the correct end of the repeat. In contrast, when the Cas1 C-terminal tail was mutated, the prespacer was integrated in a random orientation. Delayed trimming ensures preferential integration of only correctly oriented prespacers.

Through this work, Kim et al.⁹ have meticulously decoded the sequence of events during spacer acquisition. Not surprisingly, several new questions emerge. Cas1–Cas2 is known to interact with the Cascade–Cas3 effector complex.^15,16 Are ssDNA degradation products transferred directly from Cascade–Cas3 to Cas1–Cas2, creating a more efficient system for generating prespacers? It has also been speculated that adaptation might occur rapidly at Chi sites, with RecBCD generated ssDNA loops.⁷ Do these loops act as a landing platform for Cas1–Cas2 for prespacer capture? Cas4 has been shown to dictate spacer orientation in other type I systems.¹⁰ Is this due to delayed processing or through an alternative mechanism? Our new understanding of the details of type I-E adaptation bring into focus the next frontier of questions related to CRISPR-Cas adaptation.

References

Marraffini

. CRISPR-Cas immunity in prokaryotes. Nature, 2015; 526:55–61. DOI: 10.1038/nature15386.

Jackson

, McKenzie

, Fagerlund

, et al. CRISPR-Cas: adapting to change. Science, 2017; 356. DOI: 10.1126/science.aal5056.

Yosef

, Goren

, Qimron

. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res, 2012; 40:5569–5576. DOI: 10.1093/nar/gks216.

Arslan

, Hermanns

, Wurm

, et al. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system. Nucleic Acids Res, 2014; 42:7884–7893. DOI: 10.1093/nar/gku510.

Nuñez

, Kranzusch

, Noeske

, et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat Struct Mol Biol, 2014; 21:528–534. DOI: 10.1038/nsmb.2820.

Nuñez

, Lee

ASY

, Engelman

, et al. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature, 2015; 519:193–198. DOI: 10.1038/nature14237.

Levy

, Goren

, Yosef

, et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature, 2015; 520:505–510. DOI: 10.1038/nature14302.

Künne

, Kieper

, Bannenberg

, et al. Cas3-derived target DNA degradation fragments fuel primed CRISPR adaptation. Mol Cell, 2016; 63:852–864. DOI: 10.106/j.molcel.2016.07.011.

Kim

, Loeff

, Colombo

, et al. Selective loading and processing of prespacers for precise CRISPR adaptation. Nature, 2020; 579:141–145. DOI: 10.1038/s41586-020-2018-1.

10.

Shiimori

, Garrett

, Graveley

, et al. Cas4 nucleases define the PAM, length, and orientation of DNA fragments integrated at CRISPR loci. Mol Cell, 2018; 70:814–824.e6. DOI: 10.1016/j.molcel.2018.05.002.

11.

Kieper

, Almendros

, Behler

, et al. Cas4 facilitates PAM-compatible spacer selection during CRISPR adaptation. Cell Rep, 2018; 22:3377–3384. DOI: 10.1016/j.celrep.2018.02.103.

12.

Lee

, Zhou

, Taylor

, et al. Cas4-dependent prespacer processing ensures high-fidelity programming of CRISPR arrays. Mol Cell, 2018; 70:48–59.e5. DOI: 10.1016/j.molcel.2018.03.003.

13.

Lee

, Dhingra

, Sashital

. The Cas4–Cas1–Cas2 complex mediates precise prespacer processing during CRISPR adaptation. Elife, 2019; 8. DOI: 10.7554/eLife.44248.

14.

Rollie

, Graham

, Rouillon

, et al. Prespacer processing and specific integration in a Type I-A CRISPR system. Nucleic Acids Res, 2018; 46:1007–1020. DOI: 10.1093/nar/gkx1232.

15.

Redding

, Sternberg

, Marshall

, et al. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system. Cell, 2015; 163:854–865. DOI: 10.1016/j.cell.2015.10.003.

16.

Dillard

, Brown

, Johnson

, et al. Assembly and translocation of a CRISPR-Cas primed acquisition complex. Cell, 2018; 175:934–946.e15. DOI: 10.1016/j.cell.2018.09.039.