Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2022)

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.

    • Article
    Research Brief (2022)
      Cover page: The Death of Downtown? Pandemic Recovery Trajectories across 62 North American Cities
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2023)
        Cover page: Can we save the downtown? Examining pandemic recovery trajectories across 62 North American citiesCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2023)
        CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.
          Cover page: Genome expansion by a CRISPR trimmer-integrase
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2021)
          CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
            Cover page: Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complexCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2019)
            Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative β-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.
              Cover page: DABs are inorganic carbon pumps found throughout prokaryotic phyla
              Top