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1. Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria

   https://doi.org/10.1038/s42003-023-04451-8  

   Li, Chenhao ; Wang, Longfei ; Cseke, Leland J. ; Vasconcelos, Fernanda ; Huguet-Tapia, Jose Carlos ; Gassmann, Walter ; Pauwels, Laurens ; White, Frank F. ; Dong, Hansong ; Yang, Bing ( January 2023 , Communications Biology)

   Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBE_RecApis efficient in creating base edits in strains ofXanthomonas,Pseudomonas,ErwiniaandAgrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate inPseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates ofXanthomonas oryzaepv.oryzaerevealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBE_RecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

    

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2. CRISPR‐Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella ) patens

   https://doi.org/10.1002/cpz1.725  

   Wu, Shu‐Zon ; Ryken, Samantha E. ; Bezanilla, Magdalena ( April 2023 , Current Protocols)

   Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC.

   Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector

   Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors

   Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template

   Alternate Protocol 2: Designing the plasmid‐based HDR template

   Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts

   Basic Protocol 4: Identifying edited plants.

    

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3. Improved genome editing by an engineered CRISPR-Cas12a

   https://doi.org/10.1093/nar/gkac1192  

   Ma, Enbo ; Chen, Kai ; Shi, Honglue ; Stahl, Elizabeth C. ; Adler, Ben ; Trinidad, Marena ; Liu, Junjie ; Zhou, Kaihong ; Ye, Jinjuan ; Doudna, Jennifer A. ( December 2022 , Nucleic Acids Research)

   CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

    

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4. Two-metal ion mechanism of DNA cleavage in CRISPR-Cas9

   https://doi.org/10.26434/chemrxiv.9784082  

   Casalino Lorenzo, Jinek Martin ( January 2019 , ChemRxiv)

   CRISPR-Cas9 is a cutting-edge genome-editing technology, which employs the endonuclease Cas9 to cleave DNA sequences of interest. However, the catalytic mechanism of DNA cleavage and the critical role of the Mg2+ ions have remained elusive. Here, quantum–classical QM(Car-Parrinello)/MM simulations are used to disclose the two-Mg2+ aided mechanism of phosphodiester bond cleavage in the RuvC domain. We reveal that the catalysis proceeds through an associative pathway activated by H983 and fundamentally assisted by the joint dynamics of the two Mg2+ ions, which cooperatively act to properly orient the reactants and lead the chemical step to completion. Cross-validation of this mechanism is achieved by evaluating alternative reaction pathways and in light of experimental data, delivering fundamental insights on how CRISPR-Cas9 cleaves nucleic acids. This knowledge is critical for improving the Cas9 catalytic efficiency and its metal-dependent function, helping also the development of novel Cas9-based genome-editing tools. 

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5. In the business of base editors: Evolution from bench to bedside

   https://doi.org/10.1371/journal.pbio.3002071  

   Porto, Elizabeth M. ; Komor, Alexis C. ( April 2023 , PLOS Biology)

   With the advent of recombinant DNA technology in the 1970s, the idea of using gene therapies to treat human genetic diseases captured the interest and imagination of scientists around the world. Years later, enabled largely by the development of CRISPR-based genome editing tools, the field has exploded, with academic labs, startup biotechnology companies, and large pharmaceutical corporations working in concert to develop life-changing therapeutics. In this Essay, we highlight base editing technologies and their development from bench to bedside. Base editing, first reported in 2016, is capable of installing C•G to T•A and A•T to G•C point mutations, while largely circumventing some of the pitfalls of traditional CRISPR/Cas9 gene editing. Despite their youth, these technologies have been widely used by both academic labs and therapeutics-based companies. Here, we provide an overview of the mechanics of base editing and its use in clinical trials.

    

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