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1. The bridge helix of Cas12a imparts selectivity in cis ‐DNA cleavage and regulates trans ‐DNA cleavage

   https://doi.org/10.1002/1873-3468.14051  

   Parameshwaran, Hari Priya ; Babu, Kesavan ; Tran, Christine ; Guan, Kevin ; Allen, Aleique ; Kathiresan, Venkatesan ; Qin, Peter Z. ; Rajan, Rakhi ( February 2021 , FEBS Letters)

   Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity inStreptococcus pyogenes(Spy) Cas9. Here, we examined a BH variant of Cas12a fromFrancisella novicida(FnoCas12a^KD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12a^KD2Paccumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a^KD2Palso possess reducedtrans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in bothcis‐andtrans‐cleavages and show its conserved role in target discrimination among Cas nucleases.

    

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2. Novel molecular requirements for CRISPR RNA-guided transposition

   https://doi.org/10.1093/nar/gkad270  

   Walker, Matt W. G. ; Klompe, Sanne E. ; Zhang, Dennis J. ; Sternberg, Samuel H. ( April 2023 , Nucleic Acids Research)

   CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

    

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3. Gene Editing in Dimorphic Fungi Using CRISPR/Cas9

   https://doi.org/10.1002/cpmc.132  

   Kujoth, Gregory C. ; Sullivan, Thomas D. ; Klein, Bruce S. ( December 2020 , Current Protocols in Microbiology)

   Dimorphic fungi in the generaBlastomyces,Histoplasma,Coccidioides, andParacoccidioidesare important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA‐guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, withBlastomyces dermatitidisin particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9‐expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids intoBlastomycesusingAgrobacterium‐mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene‐editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors

   Support Protocol 1: Choosing protospacers in the target gene

   Basic Protocol 2:Agrobacterium‐mediated transformation ofBlastomyces

   Support Protocol 2: Preparation of electrocompetentAgrobacterium

   Support Protocol 3: Preparation and recovery ofBlastomycesfrozen stocks

    

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4. Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

   https://doi.org/10.1073/pnas.2202590119  

   Park, Jung-Un ; Tsai, Amy Wei-Lun ; Chen, Tiffany H. ; Peters, Joseph E. ; Kellogg, Elizabeth H. ( August 2022 , Proceedings of the National Academy of Sciences)

   CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB’s tetrameric architecture is stabilized by a different set of protein–protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5′ nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor–DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue “hook” that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools. 

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5. Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors

   https://doi.org/10.1093/nar/gkae024  

   Berríos, Kiara N. ; Barka, Aleksia ; Gill, Jasleen ; Serrano, Juan C. ; Bailer, Peter F. ; Parker, Jared B. ; Evitt, Niklaus H. ; Gajula, Kiran S. ; Shi, Junwei ; Kohli, Rahul M. ( January 2024 , Nucleic Acids Research)

   The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for ‘capture’ by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.

    

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