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1. A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

   https://doi.org/10.1128/AEM.01402-20  

   Dhamad, Ahmed E.; Lessner, Daniel J. (October 2020, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens. 

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2. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences

   https://doi.org/10.1038/s41556-021-00836-1  

   Wang, Chengkun; Qu, Yuanhao; Cheng, Jason K.; Hughes, Nicholas W.; Zhang, Qianhe; Wang, Mengdi; Cong, Le (February 2022, Nature Cell Biology)

   Abstract Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR–Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9–SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9–SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9–SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering. 

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3. CRISPR‐Act3.0‐Based Highly Efficient Multiplexed Gene Activation in Plants

   https://doi.org/10.1002/cpz1.365  

   Pan, Changtian; Qi, Yiping (February 2022, Current Protocols)

   Abstract CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein)‐mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double‐strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9‐mediated multiplexed gene activation system based on the CRISPR‐Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR‐Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR‐Act3.0 vectors using rice protoplasts 

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4. Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites

   https://doi.org/10.1038/s41556-022-00975-z  

   Zou, Roger S.; Marin-Gonzalez, Alberto; Liu, Yang; Liu, Hans B.; Shen, Leo; Dveirin, Rachel K.; Luo, Jay X. J.; Kalhor, Reza; Ha, Taekjip (September 2022, Nature Cell Biology)

   Abstract Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci. 

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5. A programmable DNA roadblock system using dCas9 and multivalent target sites

   https://doi.org/10.1371/journal.pone.0268099  

   Matozel, Emily K.; Parziale, Stephen; Price, Allen C. (May 2022, PLOS ONE)

   Volkert, Michael R. (Ed.)

   A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency—in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA. 

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