The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.

Xyloglucan (XG) is an abundant polysaccharide in plant cell walls and some seeds and requires at least four different enzymes for complete depolymerization. Recombinant xyloglucanase from Aspergillus niveus (XegA), α‐xylosidase from Escherichia coli (YicI), β‐galactosidase from Hypocrea jecorina (Bga1) and β‐glucosidase from H. jecorina (Bgl1) were covalently immobilized individually and in combination on chitosan‐coated ferromagnetic iron oxide nanoparticles functionalized with glutaraldehyde. All immobilized enzymes presented reduced specific catalytic activity, where immobilized YicI, XegA, Bga1 and Bgl1 retained 14.9, 76.9, 29.5 and 6.6% activity as compared with the free enzymes, respectively. Immobilized and free enzymes presented similar optimum catalytic pH and optimum catalytic temperatures differed by ±10°C between the free and immobilized enzymes. Bga1 and Bgl1 presented decreased maximum catalytic temperatures (Topt), while YicI presented an increased Topt. The Topt of XegA remained unaltered. Mass spectrometry confirmed that nanoparticles carrying all four co‐immobilized enzymes degraded XG to glucose, galactose and xylose, and higher proportions of co‐immobilized Bgl1 and XegA resulted in higher XG saccharification. Although levels of Bgl1 activity were limiting, five re‐use cycles of the co‐immobilized enzymes were demonstrated, providing proof‐of‐principle for the use of a four‐component multienzyme nanoparticle in the breakdown of a complex polysaccharide. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd.

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Xilose isomerases (XIs) são enzimas que catalisam a isomerização de D-xilose a Dxilulose in vivo, sendo também capaz de isomerizar outros açucares, como D-glicose, Dribose, L-arabinose, entre outros. A xilose isomerase de Piromyces sp. E2 (PirE2_XI) foi