Genetic Engineering Tools Research Articles

Industrial Processing of Commercially Significant Enzymes

Abstract: Enzymes are highly specific and highly sensitive biocatalyst proteins that play important roles in various life processes. As they are proteins, thus, they are composed of amino acids joined together to form chains. These amino acids are linked by peptide bonds that form between the amino-terminal and carboxylic end. There is the multifaceted application of enzymes in various sectors that include food, textile, paints, pharmaceuticals, leather, oil industries, etc. There is a wide range of sources available for the extraction of enzymes. The commercially important enzymes are generally harvested from microbial sources. Some enzymes are produced extracellularly while some enzymes are produced intracellularly. The production of this bioactive compound could be enhanced by applying suitable optimization techniques, genetic engineering tools, and other modern techniques. One of the major challenges in the commercial production of enzymes is their extraction in pure form without losing their properties. Extraction of enzymes starts from upstream processing that mainly includes two types of fermentation; one is submerged fermentation and another one is solid-state fermentation. After upstream processing, downstream processing is followed which includes cell disruption, filtration, sedimentation and centrifugation, flocculation and coagulation, and chromatography. Then, enzyme purification strategies are followed (salting-out method, dialysis, gel filtration, ion-exchange chromatography, gel electrophoresis. The purpose of down streaming is to purify and concentrate the enzyme from the complex bulk matrix. A significant amount of total production cost is contributed by downstream processing. The review discusses the industrial importance of enzymes and their downstream processing techniques economically and sustainably.

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

BackgroundDespite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming.ResultsIn this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes.ConclusionsThe novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

Advances in the Application of the Non-Conventional Yeast Pichia kudriavzevii in Food and Biotechnology Industries.

Pichia kudriavzevii is an emerging non-conventional yeast which has attracted increased attention for its application in food and biotechnology areas. It is widespread in various habitats and often occurs in the spontaneous fermentation process of traditional fermented foods and beverages. The contributions of P. kudriavzevii in degrading organic acid, releasing various hydrolase and flavor compounds, and displaying probiotic properties make it a promising starter culture in the food and feed industry. Moreover, its inherent characteristics, including high tolerance to extreme pH, high temperature, hyperosmotic stress and fermentation inhibitors, allow it the potential to address technical challenges in industrial applications. With the development of advanced genetic engineering tools and system biology techniques, P. kudriavzevii is becoming one of the most promising non-conventional yeasts. This paper systematically reviews the recent progress in the application of P. kudriavzevii to food fermentation, the feed industry, chemical biosynthesis, biocontrol and environmental engineering. In addition, safety issues and current challenges to its use are discussed.

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.

Development and Perspective of Rhodotorula toruloides as an Efficient Cell Factory.

Rhodotorula toruloides is receiving significant attention as a novel cell factory because of its high production of lipids and carotenoids, fast growth and high cell density, as well as the ability to utilize a wide variety of substrates. These attractive traits of R. toruloides make it possible to become a low-cost producer that can be engineered for the production of various fuels and chemicals. However, the lack of understanding and genetic engineering tools impedes its metabolic engineering applications. A number of research efforts have been devoted to filling these gaps. This review focuses on recent developments in genetic engineering tools, advances in systems biology for improved understandings, and emerging engineered strains for metabolic engineering applications. Finally, future trends and barriers in developing R. toruloides as a cell factory are also discussed.

Genetic manipulations in helminth parasites.

Screening of vaccine or drug target in parasitic helminth is hindered by lack of robust tool for functional studies of parasite protein which account for the availability of only a few anti-helminthic vaccines, diagnostic assay and slower pace of development of an anthelmintic drug. With the piling up of parasite transcriptomic and genomic data, in silico screening for possible vaccine/drug target could be validated by functional characterization of proteins by RNA interference or CRISPR/Cas9. These reverse genetic engineering tools have opened up a better avenue and opportunity for screening parasitic proteins in vitro as well as in vivo. RNA interference provides a technique for silencing targeted mRNA transcript for understanding a gene function in helminth as evidence by work in Caenorhabditis elegans. Recent genetic engineering tool, CRISPR/Cas9 allows knock-out/deletion of the desired gene in parasitic helminths and the other provision it provides in terms of gene knock-in/insertion in parasite genome is still to be explored in future. This manuscript discussed the work that has been carried out on RNAi and CRISPR/Cas9 for functional studies of helminth parasitic proteins.

Function of Tryptophan 2,3-Dioxygenase in Monochamus alternatus Hope Revealed by RNA Interference

Monochamus alternatus Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars of economic losses, is a serious pest of Pinus massoniana in many Asian countries. An efficient RNAi system is helpful for functional genomics research on M. alternatus. In this study, a tryptophan 2,3-dioxygenase (TDO) related to the ommochrome synthesis in insects was identified. Using RNAi technology, the M. alternatus TDO gene was silenced by injecting dsRNA into pupae, and individuals were analyzed by phenotype and expression of the TDO gene by RT-qPCR. The results show that TDO is expressed in different developmental stages of M. alternatus, having its peak expression during the prepupal stage. White-eye phenotypes were observed in the pupal and adult stages after dsRNA injection, and a significant 81% decrease in TDO mRNA levels 48 h after injection was determined by RT-qPCR. This gene can be used as a genetic marker and is an important discovery for future genetic engineering tools to control M. alternatus populations.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

Flavonoid Production: Current Trends in Plant Metabolic Engineering and De Novo Microbial Production.

Flavonoids are secondary metabolites that represent a heterogeneous family of plant polyphenolic compounds. Recent research has determined that the health benefits of fruits and vegetables, as well as the therapeutic potential of medicinal plants, are based on the presence of various bioactive natural products, including a high proportion of flavonoids. With current trends in plant metabolite research, flavonoids have become the center of attention due to their significant bioactivity associated with anti-cancer, antioxidant, anti-inflammatory, and anti-microbial activities. However, the use of traditional approaches, widely associated with the production of flavonoids, including plant extraction and chemical synthesis, has not been able to establish a scalable route for large-scale production on an industrial level. The renovation of biosynthetic pathways in plants and industrially significant microbes using advanced genetic engineering tools offers substantial promise for the exploration and scalable production of flavonoids. Recently, the co-culture engineering approach has emerged to prevail over the constraints and limitations of the conventional monoculture approach by harnessing the power of two or more strains of engineered microbes to reconstruct the target biosynthetic pathway. In this review, current perspectives on the biosynthesis and metabolic engineering of flavonoids in plants have been summarized. Special emphasis is placed on the most recent developments in the microbial production of major classes of flavonoids. Finally, we describe the recent achievements in genetic engineering for the combinatorial biosynthesis of flavonoids by reconstructing synthesis pathways in microorganisms via a co-culture strategy to obtain high amounts of specific bioactive compounds.

Synthetic activation of yeast stress response improves secretion of recombinant proteins

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies. The availability of powerful genetic engineering and synthetic biology tools has facilitated improvement of this cell factory to overcome certain limitations. However, cell engineering to improve secretion often remains a trial-and-error approach and improvements are often specific to the protein produced. Where multiple genetic interventions are needed to remove bottlenecks in the process of recombinant protein secretion, this leads to a high number of combinatorial possibilities for creation of new production strains. Therefore, our aim was to exploit whole transcriptional programs (stress response pathways) in order to simplify the strain engineering of new production strains. Indeed, the artificial activation of the general stress response transcription factor Msn4, as well as synthetic versions thereof, could replace the secretion enhancing effect of several cytosolic chaperones. Greater than 4-fold improvements in recombinant protein secretion were achieved by overexpression of MSN4 or synMSN4, either alone or in combination with Hac1 or ER chaperones. With this concept we were able to successfully engineer strains reaching titers of more than 2.5 g/L scFv and 8 g/L VHH in bioreactor cultivations. This increased secretion capacity of different industrially relevant model proteins indicates that MSN4 overexpression most likely represents a general concept to improve recombinant protein production in yeast.

Human nature transformation in the context of ecosystem recovery

The article deals with the problem of preserving human identity as a representative of Homo sapiens in the context of ecosystem recovery in the frame of the global environmental crisis deepening at the turn of the 20th and 21st centuries. It analyses different points of view in modern sciences and philosophy to the problem of human nature, as well as natural and socio-cultural factors leading to the transformation of human nature within the accelerated development of technogenic civilization. The article outlines the positive and negative trends in the socio-anthropological transformations of human nature associated with the use of the latest biotechnologies, genetic engineering tools, pharmacology, etc.

Abstract 1904: Large-scale Functional Characterization of the STIM1 Calcium Binding cEF-hand Using Novel Genetic Engineering Tools

Abstract 1904: Large-scale Functional Characterization of the STIM1 Calcium Binding cEF-hand Using Novel Genetic Engineering Tools

Determining On‐Target, Off‐Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe‐Harbor Modification of iPSCs Using Double‐Control Quantitative Copy Number PCR

Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.

Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination.

Targeted gene inactivation (TGI) is a widely used technique for the study of genes' functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR). For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing. The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted. In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria's genes.

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review.

Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.

The role of LTR retrotransposons in plant genetic engineering: how to control their transposition in the genome.

We briefly discuss that the similarity of LTR retrotransposons to retroviruses is a great opportunity for the development of a genetic engineering tool that exploits intragenic elements in the plant genome for plant genetic improvement. Long terminal repeat (LTR) retrotransposons are very similar to retroviruses but do not have the property of being infectious. While spreading between its host cells, a retrovirus inserts a DNA copy of its genome into the cells. The ability of retroviruses to cause infection with genome integration allows genes to be delivered to cells and tissues. Retrovirus vectors are, however, only specific to animals and insects, and, thus, are not relevant to plant genetic engineering. However, the similarity of LTR retrotransposons to retroviruses is an opportunity to explore the former as a tool for genetic engineering. Although recent long-read sequencing technologies have advanced the knowledge about transposable elements (TEs), the integration of TEs is still unable either to control them or to direct them to specific genomic locations. The use of existing intragenic elements to achieve the desired genome composition is better than using artificial constructs like vectors, but it is not yet clear how to control the process. Moreover, most LTR retrotransposons are inactive and unable to produce complete proteins. They are also highly mutable. In addition, it is impossible to find a full active copy of a LTR retrotransposon out of thousands of its own copies. Theoretically, if these elements were directly controlled and turned on or off using certain epigenetic mechanisms (inducing by stress or infection), LTR retrotransposons could be a great opportunity to develop a genetic engineering tool using intragenic elements in the plant genome. In this review, the recent developments in uncovering the nature of LTR retrotransposons and the possibility of using these intragenic elements as a tool for plant genetic engineering are briefly discussed.

Integrated High-Throughput Sequencing, Microarray Hybridization and Degradome Analysis Uncovers MicroRNA-Mediated Resistance Responses of Maize to Pathogen Curvularia lunata.

Curvularia lunata (Wakker) Boed, the causal agent of leaf spot in maize, is prone to mutation, making it difficult to control. RNAi technology has proven to be an important tool of genetic engineering and functional genomics aimed for crop improvement. MicroRNAs (miRNAs), which act as post-transcriptional regulators, often cause translational repression and gene silencing. In this article, four small RNA (sRNA) libraries were generated from two maize genotypes inoculated by C. lunata; among these, ltR1 and ltR2 were from the susceptible variety Huangzao 4 (HZ), ltR3 and ltR4, from the resistant variety Luyuan (LY), and 2286, 2145, 1556 and 2504 reads were annotated as miRNA in these four sRNA libraries, respectively. Through the combined analysis of high-throughput sequencing, microarray hybridization and degradome, 48 miRNAs were identified as being related to maize resistance to C. lunata. Among these, PC-732 and PC-169, two new maize miRNAs discovered, were predicted to cleave mRNAs of metacaspase 1 (AMC1) and thioredoxin family protein (Trx), respectively, possibly playing crucial roles in the resistance of maize to C. lunata. To further confirm the role of PC-732 in the interaction of maize and C. lunata, the miRNA was silenced through STTM (short tandem target mimic) technology, and we found that knocking down PC-732 decreased the susceptibility of maize to C. lunata. Precisely speaking, the target gene of PC-732 might inhibit the expression of disease resistance-related genes during the interaction between maize and C. lunata. Overall, the findings of this study indicated the existence of miRNAs involved in the resistance of maize to C. lunata and will contribute to rapidly clarify the resistant mechanism of maize to C. lunata.

Unlocking the hidden variation from wild repository for accelerating genetic gain in legumes.

The fluctuating climates, rising human population, and deteriorating arable lands necessitate sustainable crops to fulfil global food requirements. In the countryside, legumes with intriguing but enigmatic nitrogen-fixing abilities and thriving in harsh climatic conditions promise future food security. However, breaking the yield plateau and achieving higher genetic gain are the unsolved problems of legume improvement. Present study gives emphasis on 15 important legume crops, i.e., chickpea, pigeonpea, soybean, groundnut, lentil, common bean, faba bean, cowpea, lupin, pea, green gram, back gram, horse gram, moth bean, rice bean, and some forage legumes. We have given an overview of the world and India's area, production, and productivity trends for all legume crops from 1961 to 2020. Our review article investigates the importance of gene pools and wild relatives in broadening the genetic base of legumes through pre-breeding and alien gene introgression. We have also discussed the importance of integrating genomics, phenomics, speed breeding, genetic engineering and genome editing tools in legume improvement programmes. Overall, legume breeding may undergo a paradigm shift once genomics and conventional breeding are integrated in the near future.

Whole-genome sequence of the filamentous diazotrophic cyanobacterium Tolypothrix sp. PCC 7712 and its comparison with non-diazotrophic Tolypothrix sp. PCC 7601.

Cyanobacteria are highly promising microorganisms in forthcoming biotechnologies. Besides the systematic development of molecular tools for genetic engineering, the design of chassis strains and novel reactor concepts are in focus. The latter includes capillary biofilm reactors (CBR), which offer a high surface area-to-volume ratio and very high cell densities. In this context, Tolypothrix sp. PCC 7712 was found to be highly suited for this reactor system due to maximal surface coverage, extraordinarily strong biofilm attachment, and high biomass formation. Here, we provide the genome sequence of Tolypothrix sp. PCC 7712 to potentially allow targeted strain engineering. Surprisingly, it was almost identical to an available incomplete genome draft of Tolypothrix sp. PCC 7601. Thus, we completely sequenced this strain as well and compared it in detail to strain PCC 7712. Comparative genome analysis revealed 257 and 80 unique protein-coding sequences for strains PCC 7601 and PCC 7712, respectively. Clustering genomes based on average nucleotide identity (ANI) and 16S rRNA homology showed 99.98% similarity and only minor distance, respectively, between the two strains in contrast to 21 other cyanobacterial genomes. Despite these high similarities, both strains differ in the ability to fix atmospheric nitrogen and show specific sequence variations, which are discussed in the paper.

Bacteriophage genome engineering with CRISPR-Cas13a.

Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.