Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing.

In vivo gene delivery mediated by non-viral vectors for cancer therapy.

Protein/antibody therapeutics have exhibited the advantages of high specificity and activity even at an extremely low concentration compared to small molecule drugs. However, they are accompanied by unfavorable physicochemical properties such as fragile tertiary structure, large molecular size, and poor penetration of the membrane, and thus the clinical use of protein drugs is hindered by inefficient delivery of proteins into the host cells. To overcome the challenges associated with protein therapeutics and enhance their biopharmaceutical applications, various protein-loaded nanocarriers with desired functions, such as lipid nanocapsules, polymeric nanoparticles, inorganic nanoparticles, and peptides, are developed. In this review, the different strategies for intracellular delivery of proteins are comprehensively summarized. Their designed routes, mechanisms of action, and potential therapeutics in live cells or in vivo are discussed in detail. Furthermore, the perspective on the new generation of delivery systems toward the emerging area of protein-based therapeutics is presented as well.

Rational Design of Nanocarriers for Intracellular Protein Delivery.

Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazola...

Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.

Cytosolic delivery of CRISPR/Cas9 ribonucleoproteins for genome editing using chitosan-coated red fluorescent protein.

"Aptamer-locker" DNA coupling with CRISPR/Cas12a-guided biosensing for high-efficiency melamine analysis.

Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

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Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production.

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs. Jie Qiaoa, Wenqiang Lia, Siyu Lina et al. develop a cost-efficient method to prepare stable and efficient CRISPR/Cas ribonucleoprotein complexes. Using a one-step purification system with an ultra-high-affinity CL7/Im7 column they successfully harvested high yields of functional Cas9 and target-specific single guided RNAs co-expressed in Escherichia coli.

/pdf/co-expression-of-cas9-and-single-guided-rnas-in-escherichia-56j92zrvfi.pdf

Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.

https://www.mdpi.com/1420-3049/24/8/1483/pdf

Jie Qiao

Papers

Cytosolic delivery of CRISPR/Cas9 ribonucleoproteins for genome editing using chitosan-coated red fluorescent protein.

"Aptamer-locker" DNA coupling with CRISPR/Cas12a-guided biosensing for high-efficiency melamine analysis.

Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production.

Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.

A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System