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In recent years, the number of tools and techniques that enable genetic material to be added, removed or altered at specifc locations in the genome has increased signifcantly. The objective is to know the structure of genomes, the function of genes and improve gene therapy. In this work we intend to explain the functioning of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palin dromic Repeats/CRISPR associated protein 9) and the advantages that this technique may have compared to previously developed techniques, such as RNA interference (RNAi), Zinc Finger Nucleases (ZFNs) and transcription activator-like efector nucleases (TALENs) in gene and genome editing. We will start with the story of the discovery, then its biological function in the adaptive immune system of bacteria against bacteriophage attack, and ending with a description of the mechanism of action and its use in gene editing. We will also discuss other Cas enzymes with great potential for use in genome editing as an alternative to Cas9. CRISPR/Cas9 is a simple, inexpensive, and efective technique for gene editing with multiple applications from the development of functional genomics and epigenetics. This technique will, in the near future, have great applications in the development of cell models for use in medical and pharmaceutical processes, in targeted therapy, and improvement of agricultural and environmental species. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019). info:eu-repo/semantics/publishedVersion

Scientists have developed and deployed successive generations of genome engineering technologies for use in plants, including meganucleases, zinc finger nucleases, TAL effector nucleases, and CRISPR nucleases. Each of these tools has been hailed as potentially revolutionary, capable of providing more efficient and precise ways to modify plant genomes toward improving agronomic traits or making fundamental discoveries. The CRISPR nucleases, in particular, have accelerated the pace of innovation and expanded the boundaries of what is achievable within the plant research space. This review will take care to discuss current plant genome engineering technologies, covering both well-established and up-and-coming tools, as well as describe potential and real-world applications.

Plant molecular farming, that is, using plants as hosts for production of therapeutic proteins and high-value compounds, has gained substantial interest in recent years. Chloroplasts in particular are an attractive subcellular compartment for expression of foreign genes. Here, we present a new method for transgene introduction and expression in chloroplasts that, unlike classically used approaches, does not require transgene insertion into the chloroplast genome. Instead, the transgene is amplified as a physically independent entity termed a 'minichromosome'. Amplification occurs in the presence of a helper protein that initiates the replication process via recognition of specific sequences flanking the transgene, resulting in accumulation of extremely high levels of transgene DNA. Importantly, we demonstrate that such amplified transgenes serve as a template for foreign protein expression, are maintained stably during plant development and are maternally transmitted to the progeny. These findings indicate that the minichromosome-based approach is an attractive tool for transgene expression in chloroplasts and for organelle genome engineering.

Phage recombinase function unit (PRFU) plays a key role in the life cycle of phage. Repurposing this system such as lambda-Redαβ or Rac-RecET for recombineering has gained success in Escherichia coli. Previous studies have showed that most PRFUs only worked well in its native hosts but poorly in the distant species. Thus, identification of new PRFUs in specific species is necessary for the development of its corresponding genetic engineering tools. Here, we present a thorough study of PRFUs in the genomes of genus Corynebacterium. We first used a database to database searching method to facilitate accurate prediction of novel PRFUs in 423 genomes. A total number of 60 sets of unique PRFUs were identified and divided into 8 types based on evolution affinities. Recombineering ability of the 8 representative PRFUs was experimentally verified in the Corynebacterium glutamicum ATCC 13032 strain. In particular, PRFU from C. aurimucosum achieved highest efficiency in both ssDNA and dsDNA mediated recombineering, which is expected to greatly facilitate genome engineering in genus Corynebacterium. These results will provide new insights for the study and application of PRFUs. KEY POINTS: • First report of bioinformatic mining and systematic analysis of Phage recombinase function unit (PRFU) in Corynebacterium genomes. • Recombineering ability of the representative PRFUs was experimentally verified in Corynebacterium glutamicum ATCC 13032 strain. • PRFU with the highest recombineering efficiency at 10-2 magnitude was identified from Corynebacterium aurimucosum.

Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.

Common wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37 bp, containing a 13 bp core and flanking sequences of 4 bp on the left and 20 bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications.

The rapid development of the CRISPR–Cas9, –Cas12a and –Cas12b genome editing systems has greatly fuelled basic and translational plant research1–6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox. An engineered SpRY Cas9 variant enables efficient gene editing without PAM requirement in rice transgenic lines and Dahurian larch protoplasts, and its derived base editors can edit the rice genome efficiently in a PAM-less fashion too.

Bacillus subtilis has been studied for more than half a century, ever since the dawn of molecular biology, as a representative Gram-positive bacterium and cell factory. Two characteristic capacities of B. subtilis, namely its natural competence for DNA uptake and high-level enzyme secretion, have been investigated and exploited intensively during these long years. As a consequence, this bacterium has evolved into an excellent platform for synthetic biological research and development. In this review, we outline basic concepts for B. subtilis cell factory engineering, and we describe several examples of its applications in the production of proteins and high-value metabolites. In particular, we highlight engineering approaches that can make the already very efficient Bacillus protein secretion pathways even more efficient for the production of enzymes and pharmaceutical proteins. We further showcase examples of metabolic engineering in B. subtilis based on synthetic biology principles to produce various high-value or health-promoting substances, especially inositol stereoisomers. We conclude that the versatile traits of B. subtilis, combined with multi-omics approaches and rapidly developing technologies for genome engineering and high-throughput screening enable us to modify and optimize this bacterium’s metabolic circuits to deliver compounds that are needed for a green and sustainable society as well as a healthy population.

Lignin, a complex aromatic polymer, is a structural component of plant biomass. It decomposes with difficulty because of its rigidity properties, however, lignin valorization is essential for the economics of lignocellulosic biorefineries. Pseudomonas putida has been extensively investigated as a promising host strain for lignin valorizations due to intrinsic traits, such as low nutritional requirement, high tolerance to toxicity, and metabolic versatility with a wide spectrum of substrates, such as aromatic compounds. Although a naturally occurring, lignin-utilizing P. putida strain has been reported, it is necessary to engineer the genome of P. putida for efficient lignin utilization. For biological lignin valorization, the decomposition of lignin polymer to low-molecular weight compounds and transformation of lignin-derived aromatic compounds to value-added chemicals is essential. Various tools of synthetic biology have been developed for the genome engineering of P. putida; efforts in metabolic engineering have been made to expand aromatic substrate specificity and to improve productivity of value-added chemicals. Development of high-performance bio-parts and biosensors for lignin valorization has also been done. In this review, we present recent research on genome engineering tools developed for P. putida and metabolic engineering employed in P. putida to improve lignin valorization.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is one of the hallmark of biological tools, contemplated as a valid and hopeful alternatives to genome editing. Advancements in CRISPR-based technologies have empowered scientists with an editing kit that allows them to employ their knowledge for deleting, replacing and lately “Gene Surgery”, and provides unique control over genes in broad range of species, and presumably in humans. These fast-growing technologies have high strength and flexibility and are becoming an adaptable tool with implementations that are altering organism’s genome and easily used for chromatin manipulation. In addition to the popularity of CRISPR in genome engineering and modern biology, this major tool authorizes breakthrough discoveries and methodological advancements in science. As scientists are developing new types of experiments, some of the applications are raising questions about what CRISPR can enable. The results of evidence-based research strongly suggest that CRISPR is becoming a practical tool for genome-engineering and to create genetically modified eukaryotes, which is needed to establish guidelines on new regulatory concerns for scientific communities.

Arabidopsis thaliana is considered as an indispensable model system across various disciplines of modern plant biology. The short life cycle, well developed classical genetics, small genome, and a plethora of distinguishable phenotypic features make it the most preferred plant system for researchers globally. Owing to these advantages, it is highly explored to study plant-pathogen interaction, comparative omics, evolutionary plasticity, epigenetics, and recently being used for development of precision genome editing tools for plants. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein system, discovered as a part of bacterial immune system is widely used as a molecular tool for targeted genome engineering. This tool superseded two well-established genome editing platforms, zinc finger nucleases and transcription activator-like effector nucleases. Here, we have discussed CRISPR/Cas based gene editing toolbox designed, sharpened and used in A. thaliana. This review focuses on pioneering innovations in CRISPR/Cas technology and subsequent application of these tools in A. thaliana which is leading to wider application of CRISPR toolbox for basic and translational research in other plants.

Synthetic biology provides powerful tools and novel strategies for designing and modifying microorganisms to function as cell factories for biomanufacturing, which is a promising approach for realizing chemical production in a green and sustainable manner. Recent advances in genetic component design and genome engineering have enabled significant progresses in the field of synthetic biology chassis that have been developed for enzymes or biochemical production based on synthetic biology strategies, with particular reference to model microorganisms, such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Saccharomyces cerevisiae. In this review, strategies for engineering four different functional cellular modules which encompass the total process of biomanufacturing are discussed, including expanding the substrate spectrum for substrate uptake modules, refactoring biosynthetic pathways and dynamic regulation for product synthesis modules, balancing energy and redox modules, and cell membrane and cell wall engineering of product storage and secretion modules. Novel strategies of integrating and coordinating different cellular modules aided by synthetic co-culturing of multiple chassis, artificial intelligence-aided data mining for guiding strain development, and the process for designing automatic chassis development via biofoundry are expected to generate next generations of model microorganism chassis for more efficient biomanufacturing. KEY POINTS: • Engineering of functional cellular modules facilitate next generations of chassis construction. • Global optimization of biosynthesis can be improved by metabolic models. • Data-driven and automatic strain development can improve microorganism chassis construction.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Diseases caused by alterations in the DNA can be overcome by providing the cells or tissues with a functional copy of the mutated gene. The most common form of gene therapy implies adding an extra genetic unit into the cell. However, new genome engineering techniques also allow the modification or correction of the existing allele, providing new possibilities, especially for dominant diseases. Gene therapies have been tested for 30 years in thousands of clinical trials, but presently, we have only three authorised gene therapy products for the treatment of inherited diseases in European Union. Here, we describe the gene therapy alternatives already on the market in the European Union and expand the scope to some clinical trials. Additionally, we discuss the ethical and regulatory issues raised by the development of these new kinds of therapies.

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation. A method combining multiplexed genome engineering, genetic code expansion and biorthogonal chemical labeling has been developed for multiple site-specific protein labeling and used to label ribosome components at sites hard to access for FRET assays.

The Cas9 nuclease initiates double-stranded breaks at the target position in DNA, which are repaired by the intracellular restoration pathways to eliminate or insert pieces of DNA. CRISPR-Cas9 is proficient and cost-effective since cutting is guided by a piece of RNA instead of protein. Emphasis on this technology, in contrast with two recognized genome editing platforms (i.e., ZFNs and TALENs), is provided. This review evaluates the benefits of chemically synthesized gRNAs as well as the integration of chemical amendments to improve gene editing efficiencies. CRISPR is an indispensable means in biological investigations and is now as well transforming varied fields of biotechnology and agriculture. Recent advancement in targetable epigenomic-editing tools allows researchers to dispense direct functional and transcriptional significance to locus-explicit chromatin adjustments encompassing gene regulation and editing. An account of diverse sgRNA design tools is provided, principally on their target competence prediction model, off-target recognition algorithm, and generation of instructive annotations. The modern systems that have been utilized to deliver CRISPR-Cas9 in vivo and in vitro for crop improvement viz. nutritional enhancement, production of drought-tolerant and disease-resistant plants, are also highlighted. The conclusion is focused on upcoming directions, biosafety concerns, and expansive prospects of CRISPR technologies.

Muscular dystrophies (MDs) are a heterogeneous collection of inherited disorders which cause progressive muscle loss and weakness/hypotonia. Owing to the genetic root of MDs, CRISPR/Cas9 genome editing has been investigated as a possible therapy, with significant advancements having been made. This review aims to provide an overview of recent progress on the in vivo utilization of CRISPR/Cas9 in MD animal models. Three primary methods for correcting MD with CRISPR/Cas9 exist: restoration of the full-length protein, restoration of a truncated but partially functional protein, and modulation of gene expression. All these approaches have been (DMD) models with varying degrees of success. In congenital muscular dystrophy type 1A (MDC1A) mice, full-length protein restoration and disease modifier upregulation strategies significantly improved the phenotype. Lastly, efficient elimination of pathogenic CTG repeats via CRISPR/Cas9 was achieved in myotonic dystrophy type 1 (DM1) mice. Delivery of CRISPR machinery into MD animals was frequently accomplished with adeno-associated viruses (AAVs), which currently significantly outperform nanoparticle-based delivery. The targeting of satellite cells in vivo by AAVs has been evaluated by several groups in DMD mice, yielding conflicting results which require clarification. Partial or nearly complete phenotypic rescue has been achieved in DMD, MDC1A, and DM1 animals with numerous CRISPR/Cas9 strategies. While considerable work will be necessary to advance CRISPR/Cas9 genome editing past preclinical stages, its therapeutic potential for MD is extremely promising and warrants the investment.

In genome engineering, sgRNAs define the genomic target to be modified in CRISPR/Cas9 system. Either for single gene editing or genome-wide screens, sgRNAs are cloned into plasmid vectors. During our performance of CRISPR/Cas9 gene knock out, we found that the central part of a sgRNA was inverted after transformation into Escherichia coli. Interestingly, the inverted portion was found to be flanked by inverted repeats, and sealing of nicks inside the plasmid could correct the inversion. This type of sgRNA recombination completely changed its original sequence and should be noted during sgRNA design and performance of CRISPR/Cas9.

Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12b is a newly emerged genome engineering system. Here, we compared Cas12b from Alicyclobacillus acidoterrestris (Aac), Alicyclobacillus acidiphilus (Aa), Bacillus thermoamylovorans (Bth) and Bacillus hisashii (Bh) for genome engineering in rice, an important crop. We found AaCas12b was more efficient than AacCas12b and BthCas12b for targeted mutagenesis, which was further demonstrated in multiplexed genome editing. We also engineered the Cas12b systems for targeted transcriptional repression and activation. Our work establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering. The highly efficient and specific Cas12b is a new tool for genome editing with CRISPR in rice. This system can also be engineered for targeted transcriptional repression and activation.

Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C. glutamicum. Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C. glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C. glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five genes (up to ~ 13.0 kb) and knock-in of one DNA fragment. Furthermore, through kanamycin resistance repair, the ssDNA recombineering parameters were optimized. We hope the highly efficient method will be helpful for the studies of C. glutamicum, and potentially, to other bacteria. • Counterselection marker I-SceI-mediated C. glutamicum genome engineering • A suicide vector contains I-SceI expression elements and its recognition sites • Gene deletions and knock-in were conducted; efficiency was as high as 90% • Through antibiotic resistance repair, ssDNA recombineering parameters were optimized

The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1,2,3. Type I CRISPR–Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase–nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3′ end of the CRISPR RNA (crRNA). The natural Cas8–Cas5 fusion protein binds the 5′ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ–Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications., Part of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310).

Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications. Electronic supplementary material The online version of this article (10.1007/s00018-019-03396-8) contains supplementary material, which is available to authorized users.

BackgroundCO2valorization is one of the effective methods to solve current environmental and energy problems, in which microbial electrosynthesis (MES) system has proved feasible and efficient.Cupriviadus necator(Ralstonia eutropha) H16, a model chemolithoautotroph, is a microbe of choice for CO2conversion, especially with the ability to be employed in MES due to the presence of genes encoding [NiFe]-hydrogenases and all the Calvin–Benson–Basham cycle enzymes. The CO2valorization strategy will make sense because the required hydrogen can be produced from renewable electricity independently of fossil fuels.Main bodyIn this review, synthetic biology toolkit forC. necatorH16, including genetic engineering vectors, heterologous gene expression elements, platform strain and genome engineering, and transformation strategies, is firstly summarized. Then, the review discusses how to apply these tools to makeC. necatorH16 an efficient cell factory for converting CO2to value-added products, with the examples of alcohols, fatty acids, and terpenoids. The review is concluded with the limitation of current genetic tools and perspectives on the development of more efficient and convenient methods as well as the extensive applications ofC. necatorH16.ConclusionsGreat progress has been made on genetic engineering toolkit and synthetic biology applications ofC. necatorH16. Nevertheless, more efforts are expected in the near future to engineerC. necatorH16 as efficient cell factories for the conversion of CO2to value-added products.

Chromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories., Genome engineering is challenging compared to plasmid DNA manipulation. Here the authors create a simple methodology called SEGA that enables genome engineering by combining DNA and bacterial cells followed by identification of recombinant clones by a change in colour when grown on agar plates.

In this review, we have focused on the CRISPR/Cas9 technology for improving the agronomic traits in plants through point mutations, knockout, and single base editing, and we highlighted the recent progress in plant metabolic engineering. CRISPR/Cas9 technology has immense power to reproduce plants with desired characters and revolutionizing the field of genome engineering by erasing the barriers in targeted genome editing. Agriculture fields are using this advance genome editing tool to get the desired traits in the crops plants such as increase yield, improve product quality attributes, and enhance resistance against biotic and abiotic stresses by identifying and editing genes of interest. This review focuses on CRISPR/Cas-based gene knockout for trait improvement and single base editing to boost yield, quality, stress tolerance, and disease resistance traits in crops. Use of CRISPR/Cas9 system to facilitate crop domestication and hybrid breeding are also touched. We summarize recent developments and up-gradation of delivery mechanism (nanotechnology and virus particle-based delivery system) and progress in multiplex gene editing. We also shed lights in advances and challenges of engineering the important metabolic pathways that contain a variety of dietary metabolites and phytochemicals. In addition, we endorsed substantial technical hurdles and possible ways to overcome the unpredictability of CRISPR/Cas technology for broader application across various crop species. We speculated that by making a strong interconnection among all genomic fields will give a gigantic bunt of knowledge to develop crop expressing desired traits.

BackgroundGenome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs.Main bodyGenome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs.ConclusionsThis review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.

Microbial secondary metabolites are intensively explored due to their demands in pharmaceutical, agricultural and food industries. Streptomyces are one of the largest sources of secondary metabolites having diverse applications. In particular, the abundance of secondary metabolites encoding biosynthetic gene clusters and presence of wobble position in Streptomyces strains make it potential candidate as a native or heterologous host for secondary metabolite production including several cryptic gene clusters expression. Here, we have discussed the developments in Streptomyces strains genome mining, its exploration as a suitable host and application of synthetic biology for refactoring genetic systems for developing chassis for enhanced as well as novel secondary metabolites with reduced genome and cleaned background.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

Genome editing (GE) is the most powerful tool for creating genetic variation in plants. This approach is valuable for studying the mechanism of gene function and regulation as well as to improve desirable traits using sequence-specific endonucleases. It is typically performed with diverse molecular scissors that cleave a particular gene at a defined position. The advent of sequence-specific nucleases such as ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeats), in particular, have allowed for the precise and efficient introduction of genetic variation into the genome. The newly developed CRISPR-associated protein 9 (Cas9) variants, base-editing systems, novel RNA-directed nucleases, and DNA-free CRISPR/Cas9 delivery methods offer great opportunities for plant genome engineering. China has made tremendous progress in the field of GE for crop improvement to meet the demand of growing population. Herein, we reviewed the recent progress in GE of different crops in China, highlighting advanced GE tools/methods, and also discussed the specific challenges and prospects of plant GE.

The differences of DNA mutation patterns of genome editing by CRISPR (Clustered regularly interspaced Short Palindromic Repeats)-Cas9 and CRISPR-Cpf1 is not completely understood. CRISPR-Cas9 or CRISPR-Cpf1 is applied for genome editing at the exact same four genomic locations in human embryonic kidney 293T (HEK293T) cells. The mutation patterns of the target genomic loci were analyzed by targeted deep sequencing. The mutation patterns of CRISPR-Cas9 and CRISPR-Cpf1 showed prominent differences. CRISPR-Cpf1 mediated genome engineering almost always resulted in deletion, while CRISPR-Cas9 showed remarkably high rate of insertion mutation of 1 base pair. The deletion patterns of CRISPR-Cas9 and CRISPR-Cpf1 were also different. The deletion patterns of CRISPR-Cas9 were composed of diverse and evenly distributed deletion patterns, while the mutation patterns of CRISPR-Cpf1 were composed of fewer major patterns. Genome editing by CRISPR-Cas9 and CRISPR-Cpf1 shows different characteristics.

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.

In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(34) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.

In India, genetically modified organisms and products thereof are regulated under the "Rules for the manufacture, use, import, export and storage of hazardous microorganisms, genetically engineered organisms or cells, 1989" (referred to as Rules, 1989) notified under the Environment (Protection) Act, 1986. These Rules are implemented by the Ministry of Environment, Forest and Climate Change, Department of Biotechnology and State Governments though six competent authorities. The Rules, 1989 are supported by series of guidelines on contained research, biologics, confined field trials, food safety assessment, environmental risk assessment etc. The definition of genetic engineering in the Rules, 1989 implies that new genome engineering technologies including gene editing technologies like CRISPR/Cas9 and gene drives may be covered under the rules. The regulatory authorities if required, may also review the experiences of other countries in dealing with such new and emerging technologies.

The genome editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, has achieved successful therapeutic efficacy via precise modification of the genome and exceeded previous genome engineering methods owing to its versatility and simplicity. Rapid expansion in biomedical research has benefited from this newly emerged technique, such as genetic diseases treatment, cancer characterization, and plant improvement. However, the key challenge is efficient delivery of CRISPR components in vivo and nanotechnology plays an indispensable role in nonviral gene delivery. In this review, we will first briefly describe the mechanism and delivery strategies of CRISPR/Cas9 system. Furthermore, the past and current researches of nanoparticles based CRISPR/Cas9 system delivery for genome I editing will be highlighted. Finally, we will discuss the challenges and prospects of CRISPR/Cas9 system combined with nanotechnology I for clinical translation in the future.

Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.

Abiotic stresses such as extreme heat, cold, drought, and salt have brought alteration in plant growth and development, threatening crop yield and quality leading to global food insecurity. Many factors plays crucial role in regulating various plant growth and developmental processes during abiotic stresses. Ethylene response factors (ERFs) are AP2/ERF superfamily proteins belonging to the largest family of transcription factors known to participate during multiple abiotic stress tolerance such as salt, drought, heat, and cold with well-conserved DNA-binding domain. Several extensive studies were conducted on many ERF family proteins in plant species through over-expression and transgenics. However, studies on ERF family proteins with negative regulatory functions are very few. In this review article, we have summarized the mechanism and role of recently studied AP2/ERF-type transcription factors in different abiotic stress responses. We have comprehensively discussed the application of advanced ground-breaking genome engineering tool, CRISPR/Cas9, to edit specific ERFs. We have also highlighted our on-going and published R&D efforts on multiplex CRISPR/Cas9 genome editing of negative regulatory genes for multiple abiotic stress responses in plant and crop models. The overall aim of this review is to highlight the importance of CRISPR/Cas9 and ERFs in developing sustainable multiple abiotic stress tolerance in crop plants.

Multitude of gene-altering capabilities in combination with ease of design and low cost have all led to the adoption of the sophisticated and yet simple gene editing system that are clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR). The CRISPR/Cas9 system holds promise for the correction of deleterious mutations by taking advantage of the homology directed repair pathway and by supplying a correction template to the affected patient's cells. CRISPR is a tool that allows researchers to edit genes very precisely, easily and quickly. It does this by harnessing a mechanism that already existed in bacteria. Basically, there's a protein that acts like a scissors and cuts the DNA, and there's an RNA molecule that directs the scissors to any point on the genome one wants which results basically a word processor for genes. An entire gene can be taken out, put one in, or even edit just a single letter within a gene. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases, transcription activator-like effector nucleases and, most recently, CRISPR/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. However, there are still some glitches to be mended like how to regulate gene drives and its safeguards. The creation of gene knockouts is one of the first and most widely used applications of the CRISPR-Cas9 system. Nuclease-active Cas9 creates a double-strand break at the single guide RNA-targeted locus. These breaks can be repaired by homologous recombination, which can be used to introduce new mutations. When the double-strand break is repaired by the error-prone nonhomologous end joining process, indels are introduced which can produce frame shifts and stop codons, leading to functional knockout of the gene. Precedence modification have to be done on mechanism of CRISPR/Cas9, including its biochemical and structural implications incorporating the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Current applications, where the versatile CRISPR/Cas9 system is to be used to edit the genome, epigenome, or RNA of various organisms is debated. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, one should not ignore the significant ethical and biosafety concerns that it raises. Conclusively lot of prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9. Is discussed. Although many mechanistic questions remain to be answered and several challenges to be addressed yet, the use of CRISPR-Cas9-based genome technologies will increase our knowledge of disease process and their treatment in near future. Undoubtedly this field is revolutionizing in current era and may open new vistas in the treatment of fatal genetic disease.

CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics., The mechanics of the migration of T cells into tumours is an important aspect of tumour immunity. Here the authors engineer complex 3D environments to explore functions of microtubules and cell contractility as strategies to enhance T cell migration in tumour microenvironments.

Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.

During the past decade there have seen substantial progress being made on materials genome related research. However, coupling mechanisms across multi-scale microstructure and resulting consequences on property and performance of materials remain unsolved problems. Structural hierarchy, which was a concept developed but not quantitatively fulfilled in 1970s, is referred to as microstructure genome here and pinpoints the key enabler for materials genome engineering. Latest progress in deep learning for image recognition and understanding the underlying mathematical mechanisms have revealed the pivotal roles that directional wavelets and invariants play. Hierarchical invariants constructed by a wavelet system can provide an inherent descriptor for microstructure genome.

In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as “junk DNA,” have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.