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1. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R. ; Chang, Mingqin ; Cheng, Xiaohang ; Bezanilla, Magdalena ( September 2019 , Plant Direct)

   CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.

    

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2. 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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3. CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

   https://doi.org/10.1002/bit.26404  

   Schwartz, Cory ; Frogue, Keith ; Ramesh, Adithya ; Misa, Joshua ; Wheeldon, Ian ( September 2017 , Biotechnology and Bioengineering)

   In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical‐producing yeastYarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end‐joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting ofKU70andKU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR inY. lipolyticawithout permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.

    

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4. 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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5. Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines

   https://doi.org/10.1002/cpmb.129  

   Vasquez, Carlos A. ; Cowan, Quinn T. ; Komor, Alexis C. ( November 2020 , Current Protocols in Molecular Biology)

   Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Design and production of plasmids for base‐editing experiments

   Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

   Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

   Alternate Protocol 1: Next‐generation sequencing to quantify base editing

   Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

   Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

   Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

    

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