A single crRNA-tracrRNA chimera, known as a guide RNA (gRNA), can be designed for simplified use in engineered systems Prokaryotes harboring type II CRISPR/Cas systems transcribe CRISPR-RNAs (crRNAs) that hybridize with trans-activating crRNAs (tracrRNAs) that complex with the Cas9 nuclease ( Brouns et al. Modify endogenous gene regulation and epigenetic states: (TSS) transcription start site (GOI) gene of interest. ( E) Zinc finger, TALE, or deactivated nuclease-null Cas9 (dCas9) platforms can also be fused to diverse effector domains to Lead to various alterations in genomic sequence. DSB resolution through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) can ( D) Zinc fingers and TALEs fused to nuclease domains or Cas9 in complex with a gRNA can cleave targeted sequences to generateĭouble-strand breaks (DSBs). Noncomplementary strand) and genomic DNA with complementarity to the protospacer (i.e., the complementary strand), respectively The RuvC and HNH nuclease domains of Cas9 cleave genomic DNA that matches the protospacer (i.e., the The formation of this complex is dependent upon the presence of a protospacerĪdjacent motif (PAM). The protospacer segment of the gRNA and target DNA. ( C) Cas9 in complex with a chimeric guide RNA (gRNA) can recognize a specific genomic address through complementarity between ( A) and TALE repeats ( B) that recognize unique triplets or single base pairs, respectively, can be arrayed in engineered proteins to target specific Zinc finger, TALE, and Cas9-gRNA platforms for editing genomic sequence and regulatory states. Unlike ZFs and TALEs, in which protein moieties dictate DNA recognition, CRISPR/Cas systems utilize RNA-mediated Watson-Crickīonding for recognition of nucleic acids. Of bacterial and archaeal adaptive immune systems ( Barrangou et al. Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) proteins are components Similar to ZFs, individual TALE RVDs can be linked in series to localize TALEs to target loci ( Fig. Single nucleotide in target DNA ( Boch et al. TALEs consist of repeated DNA-binding domains containing repeat variable diresidues (RVDs), each of which recognizes a TALE proteins are components of plant pathogens that bind host DNA to facilitate virulence ( Kay et al. These modular ZF domains can be arrayed such that synthetic ZF DNA-binding proteins (DBPs) target a specific series ofĭNA triplets at unique genomic addresses ( Fig. Previous Section Next Section Genome engineering technologiesĬys 2-His 2 ZF domains are naturally occurring protein motifs which typically recognize three base pairs within the major groove of DNA Including synthetic zinc finger (ZF) proteins, transcription activator-like effectors (TALEs), and CRISPR/Cas9 targeting systems,Īnd their application in a new era of functional genomics. In this Perspective, we discuss the recent advances to the most commonly used genome engineering technologies, Tools are facilitating the translation of this genomic information into tangible benefits for biotechnology, agriculture,Īnd human therapeutics. Precise interrogation of the function of these genomic features and their causal role in gene regulation. The recent development of genome engineering technologies has enabled Overall cell function remain incompletely understood. However, the roles of these numerous genes, regulatory elements, epigenetic marks, and topological domains in determining 2015), as well as an understanding of genomic topological architecture ( Dekker et al. 2010 The ENCODE Project Consortium 2012 Roadmap Epigenomics Consortium et al. Recent advances have generated extensive annotation of genomic and epigenomic regulatory modules withinĬhromatin ( Bernstein et al. Genomic research has the potential to dramatically improve medicine, agriculture, biotechnology, and our fundamental understanding We also present current and potential future applications of these tools,Īs well as their current limitations and areas for future advances. Zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors forĮpigenome editing, and other emerging applications. Of the most widely adopted genome engineering platforms and their application to functional genomics. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multipleĪpproaches to functionally characterize and manipulate the regulation of genomic information. These tools can expand our understanding of fundamental biological processes and create new opportunitiesįor therapeutic designs. Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible acrossĪ variety of disciplines.
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