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Decoding CRISPR-Cas PAM recognition with UniDesign.
The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.
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Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus.
Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future.
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Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes.
A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.
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Recent Progress in CRISPR/Cas9 Technology.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9.
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TALE-based organellar genome editing and gene expression in plants.
TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.
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Engineered Nanomaterials to Potentiate CRISPR/Cas9 Gene Editing for Cancer Therapy.
Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) gene-editing technology shows promise for manipulating single or multiple tumor-associated genes and engineering immune cells to treat cancers. Currently, most gene-editing strategies rely on viral delivery; yet, while being efficient, many limitations, mainly from safety and packaging capacity considerations, hinder the use of viral CRISPR vectors in cancer therapy. In contrast, recent advances in non-viral CRISPR/Cas9 nanoformulations have paved the way for better cancer gene editing, as these nanoformulations can be engineered to improve safety, efficiency, and specificity through optimizing the packaging capacity, pharmacokinetics, and targetability. In this review, the advance in non-viral CRISPR delivery is highlighted, and there is a discussion on how these approaches can be potentially used to treat cancers in addressing the aforementioned limitations, followed by the perspectives in designing a proper CRISPR/Cas9-based cancer nanomedicine system with translational potential.
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Advances in Plant Epigenome Editing Research and Its Application in Plants.
Plant epistatic regulation is the DNA methylation, non-coding RNA regulation, and histone modification of gene sequences without altering the genome sequence, thus regulating gene expression patterns and the growth process of plants to produce heritable changes. Epistatic regulation in plants can regulate plant responses to different environmental stresses, regulate fruit growth and development, etc. Genome editing can effectively improve plant genetic efficiency by targeting the design and efficient editing of genome-specific loci with specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9). As research progresses, the CRISPR/Cas9 system has been widely used in crop breeding, gene expression, and epistatic modification due to its high editing efficiency and rapid translation of results. In this review, we summarize the recent progress of CRISPR/Cas9 in epigenome editing and look forward to the future development direction of this system in plant epigenetic modification to provide a reference for the application of CRISPR/Cas9 in genome editing.
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Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.
Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.
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Rapid improvement of domestication traits in an orphan crop by genome editing.
Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits. Thus, translating knowledge from model crops enables rapid creation of targeted allelic diversity and novel breeding germplasm in distantly related orphan crops.
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A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications.
CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.
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