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1. The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems

   https://doi.org/10.1371/journal.pone.0287461  

   Paraan, Mohammadreza; Nasef, Mohamed; Chou-Zheng, Lucy; Khweis, Sarah A.; Schoeffler, Allyn J.; Hatoum-Aslan, Asma; Stagg, Scott M.; Dunkle, Jack A. (June 2023, PLOS ONE)

   Altamirano-Bustamante, Myriam M. (Ed.)

   Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex fromS.epidermidisbound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between theS.epidermidisCas10-Csm structure and the well-resolved bacterial(S.thermophilus) and archaeal (T.onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes. 

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2. RNA targeting and cleavage by the type III-Dv CRISPR effector complex

   https://doi.org/10.1038/s41467-024-47506-y  

   Schwartz, Evan A; Bravo, Jack_P K; Ahsan, Mohd; Macias, Luis A; McCafferty, Caitlyn L; Dangerfield, Tyler L; Walker, Jada N; Brodbelt, Jennifer S; Palermo, Giulia; Fineran, Peter C; et al (December 2024, Nature Communications)

   Abstract CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction^1–5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes^6–8. Here, we determine the structures of theSynechocystistype III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors^9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes. 

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3. Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

   https://doi.org/10.3390/biology10060530  

   Thompson, Marlo K.; Sobol, Robert W.; Prakash, Aishwarya (June 2021, Biology)

   The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples. 

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4. CRISPR–Cas molecular beacons as tool for studies of assembly of CRISPR–Cas effector complexes and their interactions with DNA

   Mekler, Vladamir; Kuznedelov, Konstantin; Minakhin, Leonid; Murugan, Karthik; Sashital, Dipali G.; Severinov, Konstantin (January 2019, Methods in enzymology)

   CRISPR–Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR–Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR–Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR–Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR–Cas complexes. 

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5. Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system

   https://doi.org/10.1038/s41467-023-41802-9  

   Zhong, Zhaohui; Liu, Guanqing; Tang, Zhongjie; Xiang, Shuyue; Yang, Liang; Huang, Lan; He, Yao; Fan, Tingting; Liu, Shishi; Zheng, Xuelian; et al (December 2023, Nature Communications)

   Abstract Among CRISPR-Cas genome editing systems,Streptococcus pyogenesCas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probioticLactobacillus rhamnosus. We have confirmed the predicted 5’-NGAAA-3’ PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9’s A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond. 

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