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A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2− mutants produced in this study will be deposited in several repository organizations. Key points: • All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains • Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains • The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Currently, CRISPR/Cas9 technology has found widespread applications across various domains. However, the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety, primarily stemming from the uncontrolled activity of the system. Therefore, the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance. Biotin, characterized by its small molecular weight, and streptavidin, distinguished by its substantial spatial steric hindrance, can be harnessed as an ideal OFF switch (termed a "bioactivity brake") due to their interaction characteristics. In this work, we present a strategy that employs the streptavidin‐biotin interaction as a "brake system" for CRISPR/Cas9, effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Currently, CRISPR/Cas9 technology has found widespread applications across various domains. However, the utility of CRISPR/Cas9 is encumbered by issues pertaining to its reliability and safety, primarily stemming from the uncontrolled activity of the system. Therefore, the design and development of CRISPR/Cas9 systems with controllable activity is of paramount importance. Biotin, characterized by its small molecular weight, and streptavidin, distinguished by its substantial spatial steric hindrance, can be harnessed as an ideal OFF switch (termed a "bioactivity brake") due to their interaction characteristics. In this work, we present a strategy that employs the streptavidin‐biotin interaction as a "brake system" for CRISPR/Cas9, effectively allowing for the shutdown of the enzymatic activity of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

The GEX2 protein is expressed in the maize gamete membranes and is necessary during fertilization at the stage of contact (adhesion) of gamete membranes. Knockout of GEX2 gene can presumably lead to impaired fertilization and, as a result, to the formation of matroclinic haploid maize embryos. The purpose of this study is to analyze the efficiency of CRISPR/Cas9 editing of the GEX2 gene after PEG-mediated transfection of maize protoplasts by ribonucleoprotein (RNP) complexes with different gRNAs. For the first time, constructs for CRISPR/Cas9 editing of the maize GEX2 gene have been created, the effectiveness of which has been proven on protoplasts and reaches 10.7%, depending on the selected gRNA and the ratio and number of components in the RNP complexes. [ABSTRACT FROM AUTHOR]

Abstract Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia’s diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages in some applications. We identified 54,837 CRISPR‐Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single‐ and mixed‐species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed‐species experiments, single‐species experiments yielded more on‐target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed‐species experiments yielded sufficient data to provide ≥48‐fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL‐P6 marker. Prior work developed CRISPR‐based enrichment protocols for long‐read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short‐read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR‐based analyses of mixed‐species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori. [ABSTRACT FROM AUTHOR]

Box C/D RNAs guide the site-specific formation of 2′-O-methylated nucleotides (Nm) of RNAs in eukaryotes and archaea. Although C/D RNAs have been profiled in several archaea, their targets have not been experimentally determined. Here, we mapped Nm in rRNAs, tRNAs, and abundant small RNAs (sRNAs) and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus. The targets of C/D RNAs were assigned by analysis of base-pairing interactions, in vitro modification assays, and gene deletion experiments, revealing a complicated landscape of C/D RNA-target interactions. C/D RNAs widely use dual antisense elements to target adjacent sites in rRNAs, enhancing modification at weakly bound sites. Two consecutive sites can be guided with the same antisense element upstream of box D or D′, a phenomenon known as double-specificity that is exclusive to internal box D′ in eukaryotic C/D RNAs. Several C/D RNAs guide modification at a single non-canonical site. This study reveals the global landscape of RNA-guided 2′-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed. [ABSTRACT FROM AUTHOR]

Abstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computational techniques; limitations that may exclude field researchers, underfunded programmes and citizen scientists from contributing to cutting‐edge science. It is for these reasons that we have designed a simplified, inexpensive method for field‐based molecular organism detection—FINDeM (Field‐deployable Isothermal Nucleotide‐based Detection Method). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body‐heat inducible reactions—recombinase polymerase amplification and a CRISPR‐Cas12a fluorescent reporter assay—to amplify and detect target DNA, respectively. Here, we introduce and validate FINDeM in detecting Batrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single‐digit DNA copies from epidermal swabs in under 1 h using low‐cost supplies and field‐friendly equipment. This research signifies a breakthrough in ecology, as we demonstrate a field‐deployable platform that requires only basic supplies (i.e. micropipettes, plastic consumables and a UV flashlight), inexpensive reagents (~$1.29 USD/sample) and emanated body heat for highly sensitive, DNA‐based organism detection. By presenting FINDeM in an ecological system with pressing, global biodiversity implications, we aim to not only highlight how CRISPR‐based applications promise to revolutionize organism detection but also how the continued development of such techniques will allow for additional, more diversely trained researchers to answer the most pressing questions in ecology.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5–7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-$\Delta $32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-$\Delta $32 mutation in haematopoietic stem cells.

Abstract—It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A G and C T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine. [ABSTRACT FROM AUTHOR]

RNA has received enormous attention as a biomolecule in the fields of medicine, public health, and biotechnology. Over the last decade, various types of RNA molecules have been exploited as valuable drug targets by different classes of pharmaceutical agents in combating infections and diseases. In this work, we have investigated the binding of Cisplatin to RNA within double stranded RNA : DNA hybrid structures. UV melting and Isothermal Titration Calorimetry studies reveal that Cisplatin binding leads to destabilization of hybrid duplex likely due to Cisplatin‐RNA adduct formation. Gel electrophoretic analysis using an in vitro CRISPR‐Cas9 model system shows that Cisplatin binding to guide RNA leads to a concentration‐dependent reduction in target DNA cleavage. [ABSTRACT FROM AUTHOR]

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This dataset contains the gene sequences of the small and large sub-unit of the hydrogenase enzyme obtained from the annotated genome of Rhodobacter johrii MAY2. The whole genome sequence of the isolate was performed using SEED genome viewer on the Rapid Annotation using the Subsystem Technology (RAST) platform. Concurrently, guide RNA sequences and primers were meticulously crafted using the CHOPCHOP v.3.0 web tool, specifically designed for the precise editing and amplification of the target genes. The primers were optimized via gradient PCR to determine appropriate amplification conditions. Furthermore, the guide RNA was tested via in-vitro cleavage assay, gauging its efficacy in cleaving the intended target genes. The dataset, including the optimization and the cleavage assay, was deposited in Mendeley Data with DOI no: 10.17632/rcx3mcssnx.2.

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Background & Objective: Medulloblastoma formation is importantly related to granular cell proliferation and differentiation, processes which are under the influence of sonic hedgehog (SHH) signaling. Exons of genes encoding different components of this signaling pathway (e.g., ligands, co-receptor, transcription factors, and target genes) were investigated to identify the proper single guide RNAs for selective targeting of positive regulators of the SHH pathway. Materials & Methods: The genomic DNA sequences of corresponding genes of several positive regulators of the SHH pathway (in Homo Sapiens), including SHH, SMO, GLI1, GLI2, MYCN, and MYC, were retrieved from the National Center for Biotechnology Information (NCBI) gene database. Next, the exon sequences of these genes were identified using protospacer adjacent motif (PAM) of NGG and Streptococcus pyogenes Cas9 nuclease and evaluated by CRISPOR software to select the best sgRNA for each target gene. Results: The analyses revealed the best sgRNAs for the SHH, SMO, GLI1, GLI2, MYCN, and MYC genes targeted exon 1, -4, -4, -5, -2 and -2, respectively. Proper sgRNAs for targeting each exon in each gene were also identified. Conclusion: This study revealed possible specific exonic targets of components of the SHH signaling pathway through designing proper sgRNAs using the CRISPR/Cas9 genome editing approach. [ABSTRACT FROM AUTHOR]

Main conclusion: The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR–Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA–gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants. [ABSTRACT FROM AUTHOR]

Viral‐mediated delivery of the CRISPR‐Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane‐bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single‐cell clones. Moreover, viral‐mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non‐viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all‐in‐one mammalian CRISPR‐Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N‐acetyltransferase, allowing for transient, expression‐dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4–6 folds. Altogether ptARgenOM provides a simple, fast, and cost‐effective delivery tool for genome editing. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is rising to dangerously high levels throughout the world. To cope with this problem, scientists are working on CRISPR-based research so that antibiotic-resistant bacteria can be killed and attacked almost as quickly as antibiotic-sensitive bacteria. Nuclease activity is found in Cas9, which can be programmed with a specific target sequence. This mechanism will only attack pathogens in the microbiota while preserving commensal bacteria. This article portrays the delivery methods used in the CRISPR-Cas system, which are both viral and non-viral, along with its implications and challenges, such as microbial dysbiosis, off-target effects, and failure to counteract intracellular infections. CRISPR-based systems have a lot of applications, such as correcting mutations, developing diagnostics for infectious diseases, improving crops productions, improving breeding techniques, etc. In the future, CRISPR-based systems will revolutionize the world by curing diseases, improving agriculture, and repairing genetic disorders. Though all the drawbacks of the technology, CRISPR carries great potential; thus, the modification and consideration of some aspects could result in a mind-blowing technique to attain all the applications listed and present a game-changing potential. [ABSTRACT FROM AUTHOR]

Zeugodacus cucurbitae (Coquillett) is a major pest affecting cucurbit crops. CRISPR/Cas9 mediated mutagenesis of target genes in Z. cucurbitae by generating a series of frame-shift mutation will help in changing physiology and behaviour of the insect. White gene is an important eye pigmentation gene widely used as marker gene in Drosophila melanogaster. PCR and Cloning of Z. cucurbitae white gene (2051bp) was performed. Off-target minimized gRNAs were designed by using bioinformatics online software CHOPCHOP by giving white coding sequences as input. Efficiency of the designed sgRNA was confirmed by in vitro restriction assay. [ABSTRACT FROM AUTHOR]

In genetic engineering, the revolutionary CRISPR-Cas system has proven to be a vital tool for precise genome editing. Simultaneously, the emergence and rapid evolution of deep learning methodologies has provided an impetus to the scientific exploration of genomic data. These concurrent advancements mandate regular investigation of the state-of-the-art, particularly given the pace of recent developments. This review focuses on the significant progress achieved during 2019–2023 in the utilization of deep learning for predicting guide RNA (gRNA) activity in the CRISPR-Cas system, a key element determining the effectiveness and specificity of genome editing procedures. In this paper, an analytical overview of contemporary research is provided, with emphasis placed on the amalgamation of artificial intelligence and genetic engineering. The importance of our review is underscored by the necessity to comprehend the rapidly evolving deep learning methodologies and their potential impact on the effectiveness of the CRISPR-Cas system. By analyzing recent literature, this review highlights the achievements and emerging trends in the integration of deep learning with the CRISPR-Cas systems, thus contributing to the future direction of this essential interdisciplinary research area.

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing. A new plasmid for easy and fast CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5–7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5- $\Delta $ Δ 32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5- $\Delta $ Δ 32 mutation in haematopoietic stem cells. [ABSTRACT FROM AUTHOR]

The kinetoplastids are unicellular flagellates that derive their name from the ‘kinetoplast’, a region within their single mitochondrion harboring its organellar genome of high DNA content, called kinetoplast (k) DNA. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through RNA editing, whereby small regulatory guide (g)RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the kDNA expansion and evolution of their unique uridine insertion/deletion editing across kinetoplastids has been difficult to achieve. Here, we resolved the kDNA structure and editing patterns in the early-branching kinetoplastid Trypanoplasma borreli and compare them with those of the well-studied trypanosomatids. We find that its kDNA consists of circular molecules of about 42 kb that harbor the rRNA and protein-coding genes, and 17 different contigs of approximately 70 kb carrying an average of 23 putative gRNA loci per contig. These contigs may be linear molecules, as they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of four edited mRNAs, including one coding for ATP synthase 6 that was previously thought to be missing. We utilized computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with inconsistent editing patterns, apparently products of non-canonical editing. This species utilizes the most extensive uridine deletion compared to other studied kinetoplastids to enforce amino acid conservation of cryptogene products, although insertions still remain more frequent. Finally, in three tested mitochondrial transcriptomes of kinetoplastids, uridine deletions are more common in the raw mitochondrial reads than aligned to the fully edited, translationally competent mRNAs. We conclude that the organization of kDNA across known kinetoplastids represents variations on partitioned coding and repetitive regions of circular molecules encoding mRNAs and rRNAs, while gRNA loci are positioned on a highly unstable population of molecules that differ in relative abundance across strains. Likewise, while all kinetoplastids possess conserved machinery performing RNA editing of the uridine insertion/deletion type, its output parameters are species-specific.

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato. Key message: Potato lines, with a single or four copies of the gfp transgene integrated, were subjected to CRISPR/Cas9-mediated mutations. The results indicated that it should be possible to target and knock out multiple copies of a native gene in the tetraploid genotypes of this crop. [ABSTRACT FROM AUTHOR]

Genetically modifying mice traditionally involved complex methods of designing and validating targeting constructs, embryonic stem cell electroporation and selection, blastocyst injection, and breeding chimeras for germline transmission. Such arduous steps were best carried out by specialized gene targeting cores in academia or through expensive commercial vendors. Further, the time from initiation to completion of a project often took at least 1 year and, in some cases, much longer (or never), with no guarantees of success. The RNA-programmable CRISPR system of gene editing has greatly streamlined the generation of gene modifications (e.g., small substitutions, insertions, and deletions) in the mouse with high rates of success. Several editing platforms exist for gene/genome targeting in mice and other animal models previously difficult or impossible to alter. Here, we provide a simplified method of generating genetically modified mice using the prime editing platform. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design, cloning, and synthesis of engineered pegRNA (epegRNA) Basic Protocol 2: Microinjection of PE2 components into mouse zygote Basic Protocol 3: Genotyping founder mice and breeding for germline transmission., (© 2024 Wiley Periodicals LLC.)

CRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target-specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably in a spacer sequence-dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effector's activity and/or potential for off-target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, the study presents compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off-target combinations simultaneously in vitro for both CRISPR effector activity and specificity by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water-in-oil microemulsions. With CCR, large numbers of the candidate gRNAs (output by computational design tools) can be immediately validated in parallel, and the study shows that CCR can be used to screen hundreds of thousands of extended gRNA (x-gRNAs) variants that can completely block cleavage at off-target sequences while maintaining high levels of on-target activity. It is expected that CCR can help to streamline the gRNA generation and validation processes for applications in biological and biomedical research., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Globally, it is estimated that 43 million people are living with human immunodeficiency virus type 1 (HIV-1), and there are more than 600,000 acquired immunodeficiency syndrome (AIDS)-related deaths in 2020. The only way to increase the life expectancy of these patients right now is to use combination antiretroviral therapy (cART) for the lifetime. Due to the integration of the HIV-1 DNA in lymphocytes, the replication of the virus can only be reduced by using antiretroviral drugs. If the drug is stopped, the virus will replicate and reduce the number of lymphocytes. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9-mediated genome editing system has been considered, preventing HIV-1 replication by causing DNA double-stranded breaks (DSBs) or disrupting the integrated virus replication by targeting the provirus. In this study, we utilized the CRISPR/Cas9 without the nuclear localization signal sequence (w/o NLS) system to inhibit the VSV-G-pseudotyped HIV-1 replication by targeting the HIV-1 DNA as a prophylactic method. To this end, we designed a multiplex gRNA (guide RNA) cassette to target the pol, env, and nef/long terminal repeat (nef/LTR) regions of the HIV-1 genome and then cloned it in plasmid expressing no-NLS-Cas9 protein as an all-in-one CRISPR/Cas9 vector. Using HIV-1 pseudovirus transduction into HEK-293T cell lines, our results showed that the CRISPR/Cas9-no NLS system disrupts the pseudotyped HIV-1 DNA and significantly (P-value < 0.0001) decreases the p24 antigen shedding and viral RNA load in cell culture supernatants harvested 48h after virus transduction. Although these results revealed the potential of the CRISPR/Cas9-no NLS nuclease system as a prophylactic strategy against HIV-1 infections, due to inefficient impairments of HIV-1 DNA, further studies are required to enhance its effectiveness and application in clinical practice.

Antibiotic resistance is rising to dangerously high levels throughout the world. To cope with this problem, scientists are working on CRISPR-based research so that antibiotic-resistant bacteria can be killed and attacked almost as quickly as antibiotic-sensitive bacteria. Nuclease activity is found in Cas9, which can be programmed with a specific target sequence. This mechanism will only attack pathogens in the microbiota while preserving commensal bacteria. This article portrays the delivery methods used in the CRISPR-Cas system, which are both viral and non-viral, along with its implications and challenges, such as microbial dysbiosis, off-target effects, and failure to counteract intracellular infections. CRISPR-based systems have a lot of applications, such as correcting mutations, developing diagnostics for infectious diseases, improving crops productions, improving breeding techniques, etc. In the future, CRISPR-based systems will revolutionize the world by curing diseases, improving agriculture, and repairing genetic disorders. Though all the drawbacks of the technology, CRISPR carries great potential; thus, the modification and consideration of some aspects could result in a mind-blowing technique to attain all the applications listed and present a game-changing potential.

The efficiency of the CRISPR-Cas system is highly dependent on well-designed CRISPR RNA (crRNA). To facilitate the use of various types of CRISPR-Cas systems, there is a need for the development of computational tools to design crRNAs which cover different CRISPR-Cas systems with off-target analysis capability. Numerous crRNA design tools have been developed, but nearly all of them are dedicated to design crRNA for genome editing. Hence, we developed a tool matching the needs of both beginners and experts, named CaSilico, which was inspired by the limitations of the current crRNA design tools for designing crRNAs for Cas12, Cas13, and Cas14 CRISPR-Cas systems. This tool considers a comprehensive list of the principal rules that are not yet well described to design crRNA for these types. Using a list of important features such as mismatch tolerance rules, self-complementarity, GC content, frequency of cleaving base around the target site, target accessibility, and PFS (protospacer flanking site) or PAM (protospacer adjacent motif) requirement, CaSilico searches all potential crRNAs in a user-input sequence. Considering these features help users to rank all crRNAs for a sequence and make an informed decision about whether a crRNA is suited for an experiment or not. Our tool is sufficiently flexible to tune some key parameters governing the design of crRNA and identification of off-targets, which can lead to an increase in the chances of successful CRISPR-Cas experiments. CaSilico outperforms previous crRNA design tools in the following aspects: 1) supporting any reference genome/gene/transcriptome for which an FASTA file is available; 2) designing crRNAs that simultaneously target multiple sequences through conserved region detection among a set of sequences; 3) considering new CRISPR-Cas subtypes; and 4) reporting a list of different features for each candidate crRNA, which can help the user to select the best one. Given these capabilities, CaSilico addresses end-user concerns arising from the use of sophisticated bioinformatics algorithms and has a wide range of potential research applications in different areas, especially in the design of crRNA for pathogen diagnosis. CaSilico was successfully applied to design crRNAs for different genes in the SARS-CoV-2 genome, as some of the crRNAs have been experimentally tested in the previous studies.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications. [Display omitted] • Engineered IscB and ωRNA significantly increase programmable DNA cleavage activity • Fusion of DNA deaminases with eIscB-D efficiently induces base conversions • eIscB-D/eωRNA enables robust DNA editing in mouse embryos to generate disease models Xue et al. develop a highly efficient, miniature genome-editing system through extensive engineering of ωRNA and IscB nuclease via amino acid substitutions and HMG-D domain fusion. eIscB-D can be adapted to base editors to achieve robust A-to-G or C-to-T conversion, and it is highly efficient in mouse embryos to generate disease models. [ABSTRACT FROM AUTHOR]

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a novel molecular tool. In recent days, it has been highlighted a lot, as the Nobel prize was awarded for this sector in 2020, and also for its recent use in Covid-19 related diagnostics. Otherwise, it is an eminent gene-editing technique applied in diverse medical zones of therapeutics in genetic diseases, hematological diseases, infectious diseases, etc., research related to molecular biology, cancer, hereditary diseases, immune and inflammatory diseases, etc., diagnostics related to infectious diseases like viral hemorrhagic fevers, Covid-19, etc. In this review, its discovery, working mechanisms, challenges while handling the technique, recent advancements, applications, alternatives have been discussed. It is a cheaper, faster technique revolutionizing the medicinal field right now. However, their off-target effects and difficulties in delivery into the desired cells make CRISPR, not easily utilizable. We conclude that further robust research in this field may promise many interesting, useful results. – CRISPR has revolutionized genetics. – CRISPR was nameless till its discovery in bacteria, and now it is in the spotlight because of its award for Nobel prize 2020 as well as its role in Covid-19 related diagnostics. – It has reached every door of therapeutics, research, and diagnostics related to mankind and also plant biology. – It is essential to consider off-target effects and adverse immune reactions linked with CRISPR. – Fine-tuning of the methodology and new additions like CRISPR GUARD, multiplexing, etc., have made CRISPR more precise and straightforward. – Anyway, there is a demand for further technical modernization to reinvigorate the methodology more advanced. [ABSTRACT FROM AUTHOR]

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on Saccharomyces cerevisiae. We describe the tools and methods that have been previously developed for genome editing in Saccharomyces cerevisiae and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.

Small, noncoding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs (gRNAs) that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5' triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3' processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3'-5' exonuclease, and three additional subunits. This complex, termed mitochondrial 3' processome (MPsome), is responsible for primary uridylation of ∼800 nt gRNA precursors, their processive degradation to a mature size of 40-60 nt, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3'-5' degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5' extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3'-5' exonucleolytic degradation.

Mass spectrometry (MS) has long been used for quality control of oligonucleotide therapeutics, including single-guide RNAs (sgRNAs) for clustered regularly interspaced short palindromic repeats techniques. However, the application of MS is limited to qualitative assays in most cases. Here, we showed that electrospray-ionization quadrupole time-of-flight MS (ESI-QTOF-MS) assays can be quantitative for chemical species found in sgRNA samples. More specifically, using a 100-nt SpCas9 sgRNA as the example, we estimated that the limits of quantification for length variants in the range of N - 4 to N + 4 (i.e., 96-104 nucleotides) were equal to or lower than 1%. Our study highlighted the potential of ESI-QTOF in its application as a quality control method for sgRNA molecules.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state‐of‐the‐art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR‐associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing. [ABSTRACT FROM AUTHOR]

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world's most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops. [ABSTRACT FROM AUTHOR]

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world’s most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.