Srdx dna core recognition4/5/2023 ![]() ![]() ![]() 11, 14, 20 Other studies have also shown that the fusion of dCas9 to histone acetyltransferase was used to initiate downstream gene transcription and epigenetic modulation. 23, 24 Fusion of dCas9 to FokI endonuclease forms a synthetic fdCas9 chimeric protein that is capable of mediating efficient genome editing with improved specificity in mammalian cells compared to native Cas9. 22, 23 Domains including regulatory effectors have been used for fusion to dCas9, such as the transcriptional repressor SRDX or the transcriptional activators EDLL and TAL domains, and controlled gene expression in plants has been reported. The vast applications being continuously discovered for DNA targeting by dCas9 utilize the enzyme's adjustability to be fused to other functional domains. 9 This dCas9 variant is capable of DNA targeting but lacks the DNA cleavage function and is thus being used for other applications. ![]() 2, 7, 8 In contrast, a dead Cas9 (dCas9) exists that is derived from the inactivation of both the HNH and the RuvC nuclease domains. Active Cas9 generates blunt double-stranded DNA breaks (DSBs) using two endonuclease domains-HNH and RuvC-that create the DNA breaks at the target site guided by single-guide RNA (sgRNA). 1–6 As a form of CRISPR class 2 system, RNA-guided CRISPR-Cas9 gene editing activity is performed by the endonuclease Cas9 protein. The CRISPR-Cas system has been rapidly harnessed to perform various genomic engineering tasks. As such, we exploited this novel finding as a method to demonstrate that inserts can be ligated into vectors, and vice versa, whereby selective RE sites are temporarily sheltered to allow the desired cloning. Furthermore, we show that multiple dRNPs can be used simultaneously to inhibit more than one RE sites. We show that the inhibition of RE activities occurs when the recognition or the cleavage site of the DNA is overlapped by the sgRNA or the protospacer adjacent motif sequence. Here, we show that dCas9 and single-guide RNA preassembled to form ribonucleoprotein dCas9–sgRNA (referred to as dRNP) is capable of specifically and reversibly blocking the activity of DNA cleavage by restriction enzymes (REs). Diverse dCas9 tools have been developed for transcription regulation, epigenetic engineering, base editing, genome imaging, genetic screens, and chromatin immunoprecipitation. The creation of the nuclease-dead Cas protein (dCas9) offers a new platform for a plethora of new discoveries. ![]()
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