Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.
- Keywords
- CRISPR-Cas, Candida albicans/glabrata, Expression optimization, Metabolic engineering, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Synthetic biology, Yarrowia lipolytica, Yeasts,
- MeSH
- Point Mutation MeSH
- Chromosomes, Fungal MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing methods MeSH
- Microorganisms, Genetically-Modified MeSH
- Cloning, Molecular MeSH
- Yeasts genetics MeSH
- Metabolic Engineering MeSH
- Pichia genetics MeSH
- Gene Expression Regulation, Fungal MeSH
- Saccharomyces cerevisiae genetics MeSH
- Gene Drive Technology MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Yarrowia genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) are the only known adaptive immune system in prokaryotes. CRISPR-Cas system provides sequence-specific immunity against invasion by foreign genetic elements. It carries out its functions by incorporating a small part of the invading DNA sequence, termed as spacer into the CRISPR array. Although the CRISPR-Cas systems are mainly responsible for adaptive immune functions, their alternative role in the gene regulation, bacterial pathophysiology, virulence, and evolution has started to unravel. In several species, these systems are revealed to regulate the processes beyond adaptive immunity by employing various components of CRISPR-Cas machinery, independently or in combination. The molecular mechanisms entailing the regulatory processes are not clear in most of the instances. In this review, we have discussed some well-known and some recently established noncanonical functions of CRISPR-Cas system and its fast-extending applications in other biological processes.
According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.
- Keywords
- CRISPR/Cas9, agricultural production, genome editing, industrial applications, livestock, therapeutics,
- MeSH
- Archaea metabolism MeSH
- Bacteria metabolism MeSH
- CRISPR-Cas Systems * MeSH
- History, 20th Century MeSH
- History, 21st Century MeSH
- Livestock MeSH
- Gene Editing methods MeSH
- Genetic Engineering history methods MeSH
- Genome MeSH
- Humans MeSH
- Clustered Regularly Interspaced Short Palindromic Repeats MeSH
- Crops, Agricultural genetics MeSH
- Animals MeSH
- Check Tag
- History, 20th Century MeSH
- History, 21st Century MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Historical Article MeSH
- Review MeSH
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
- MeSH
- Bacteria enzymology genetics MeSH
- Bacteroidetes enzymology genetics MeSH
- Bacterial Proteins genetics metabolism MeSH
- CRISPR-Associated Proteins metabolism MeSH
- CRISPR-Cas Systems * MeSH
- Dimerization MeSH
- DNA Primers biosynthesis MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA Primase genetics metabolism MeSH
- Escherichia coli metabolism MeSH
- Gene Expression MeSH
- Phylogeny MeSH
- Mutation MeSH
- Prokaryotic Cells metabolism MeSH
- Recombinant Proteins MeSH
- Ribonucleotides metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Bacterial Proteins MeSH
- CRISPR-Associated Proteins MeSH
- DNA Primers MeSH
- DNA-Directed DNA Polymerase MeSH
- DNA Primase MeSH
- Recombinant Proteins MeSH
- Ribonucleotides MeSH
The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
- Keywords
- CA18111, CRISPR applications, CRISPR platforms, PlantEd, gene regulation, mutant detection, plant regeneration,
- MeSH
- CRISPR-Cas Systems * genetics MeSH
- Gene Editing * MeSH
- Plants, Genetically Modified genetics MeSH
- Genome, Plant genetics MeSH
- Plant Breeding MeSH
- Crops, Agricultural genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.
- Keywords
- Aspergillus oryzae, CRISPR/Cpf1 system, Filamentous fungi, Multiplex gene editing,
- MeSH
- Aspergillus oryzae * genetics MeSH
- Gene Editing MeSH
- Francisella * MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.
- Keywords
- CRISPR/Cas9, Giardia, gene knockout, multiploid,
- MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing methods MeSH
- Giardia lamblia * genetics MeSH
- Humans MeSH
- Tetraploidy MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
- Keywords
- COVID-19, CRISPR, Cas12b, Diagnostics, Dual-detection, One-pot detection, RT-LAMP, SARS-CoV-2, Thermophillic, Variants of concern,
- MeSH
- Brevibacillus * genetics MeSH
- COVID-19 * diagnosis MeSH
- Humans MeSH
- SARS-CoV-2 genetics MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.
- Keywords
- CRISPR/Cas9, agronomic trait improvement, cereals, multiplex editing, strategies for gain-of-function,
- MeSH
- CRISPR-Cas Systems * genetics MeSH
- Genome, Plant genetics MeSH
- Edible Grain * genetics MeSH
- Plant Breeding MeSH
- Artificial Intelligence MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Crops, Agricultural genetics MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
Target-AID, BE3, and ABE7.10 base editors fused to the catalytically modified Cas9 and xCas9(3.7) were tested for germline editing of the fruit fly Drosophila melanogaster. We developed a guide RNA-expressing construct, white-4gRNA, targeting splice sites in the white gene, an X-chromosome located gene. Using white-4gRNA flies and transgenic lines expressing Target-AID, BE3, and ABE7.10 base editors, we tested the efficiency of stable germline gene editing at three different temperatures. Classical Cas9 generating insertions/deletions by non-homologous end joining served as a reference. Our data indicate that gene editing is most efficient at 28°C, the highest temperature suitable for fruit flies. Finally, we created a new allele of the core circadian clock gene timeless using Target-AID. This base edited mutant allele timSS308-9FL had a disrupted circadian clock with a period of ∼29 h. The white-4gRNA expressing fly can be used to test new generations of base editors for future applications in Drosophila.
- MeSH
- Adenine MeSH
- CRISPR-Cas Systems * genetics MeSH
- Cytosine MeSH
- Drosophila melanogaster genetics MeSH
- Drosophila genetics MeSH
- Gene Editing * MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Germ Cells MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenine MeSH
- Cytosine MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
