Characterizing a thermostable Cas9 for bacterial genome editing and silencing

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

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Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

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A portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus paracasei.

Applied and Environmental Microbiology ◽

10.1128/aem.02669-20 ◽

2021 ◽

Author(s):

Yong Jun Goh ◽

Rodolphe Barrangou

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Genome Editing ◽

Lactobacillus Acidophilus ◽

Genome Engineering ◽

Gene Deletion ◽

Lactobacillus Paracasei ◽

Universal System ◽

Lactobacillus Gasseri ◽

Health Promoting

Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplements industries, and specific strains such as Lactobacillus acidophilus NCFM have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for customized sgRNA and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS and ltaS), yielding 35-100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted a mCherry fluorescent protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate concurrent single base substitution and gene deletion in Lactobacillus gasseri ATCC 33323, and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli, and open new avenues for the engineering of health-promoting lactic acid bacteria. Importance This work describes the development of a broad-host range CRISPR-based editing system for genome manipulations in three Lactobacillus species, which belong to lactic acid bacteria (LAB) commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiota, and their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus, and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems, and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

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Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

Microbial Cell Factories ◽

10.1186/s12934-019-1219-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Wei Shen ◽

Jun Zhang ◽

Binan Geng ◽

Mengyue Qiu ◽

Mimi Hu ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Zymomonas Mobilis ◽

Genome Engineering ◽

Gene Deletion ◽

Genetic Manipulation ◽

Strain Improvement ◽

Genomic Research ◽

Francisella Novicida ◽

A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Recent advances of Cas12a applications in bacteria

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11243-9 ◽

2021 ◽

Author(s):

Meliawati Meliawati ◽

Christoph Schilling ◽

Jochen Schmid

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Bacterial Genome ◽

Adaptive Immune System ◽

Eukaryotic Cell ◽

Promising Alternative ◽

Guide Rna ◽

Recent Advances ◽

Versatile Tool

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

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Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

10.1101/834218 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alberto Jiménez ◽

Birgit Hoff ◽

José Luis Revuelta

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Gene Deletion ◽

Marker Genes ◽

Target Sequence ◽

Ashbya Gossypii ◽

Nucleotide Substitutions ◽

Large Deletions ◽

Auxotrophic Marker ◽

Selection Of

AbstractThe CRISPR/Cas technologies constitute an essential tool for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables the efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained to the existence of an specific 5’-NGG-3’ PAM sequence in the target site.Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows to employ a T-rich PAM sequence (5’-TTTN-3’) and facilitates the implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions into five different auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system was demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may significantly affect to the edition efficiency of the system.

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A thermostable Cas9 with increased lifetime in human plasma

10.1101/138867 ◽

2017 ◽

Cited By ~ 2

Author(s):

Lucas B. Harrington ◽

David Paez-Espino ◽

Janice S. Chen ◽

Enbo Ma ◽

Brett T. Staahl ◽

...

Keyword(s):

Human Plasma ◽

Dna Cleavage ◽

Elevated Temperatures ◽

Thermophilic Bacterium ◽

Temperature Range ◽

Cas9 Protein ◽

Wide Range ◽

Rnp Complex ◽

Mammalian Genomes

CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to proteolytic degradation and unsuitable for applications requiring function at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacteriumGeobacillus stearothermophilus(GeoCas9) catalyzes RNA-guided DNA cleavage over a wide temperature range and has an enhanced protein lifetime in human plasma. GeoCas9 is active at temperatures up to 70°C, compared to 45°C forStreptococcus pyogenesCas9 (SpyCas9), which greatly expands the temperature range for CRISPR-Cas9 applications. By comparing features of two closely relatedGeobacillushomologs, we created a variant of GeoCas9 that doubles the DNA target sequences that can be recognized by this system. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP deliveryin vivoand expands the temperature range of CRISPR-Cas9.

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CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

10.1101/2020.03.23.003368 ◽

2020 ◽

Author(s):

Zhanqi Dong ◽

Qi Qin ◽

Zhigang Hu ◽

Xinling Zhang ◽

Jianghao Miao ◽

...

Keyword(s):

Antiviral Therapy ◽

Genome Editing ◽

Genome Engineering ◽

Transgenic Silkworm ◽

Editing Efficiency ◽

Transgenic Lines ◽

Transgenic Silkworms ◽

Bmnpv Genome

AbstractCRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study in insect. We compared three Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the gene editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCpf1 system in transgenic silkworm. This study demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in the silkworm, and illustrates the use of this technology for increasing disease resistance.Author SummaryGenome editing is a powerful tool that has been widely used in gene function, gene therapy, pest control, and disease-resistant engineering in most parts of pathogens research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral therapy of transgenic silkworm have emerged. Nevertheless, there is still room to expand the scope of genome editing tool for further application to improve antiviral research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency editing BmNPV genome in vitro and in vivo for the first time. More importantly, this Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare with Cas9 system, and no significant cocoons difference was observed between transgenic lines infected with BmNPV and control. These broaden the range of application of CRISPR for novel genome editing methods in silkworm and also enable sheds light on antiviral therapy.

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One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

10.21203/rs.3.rs-306012/v1 ◽

2021 ◽

Author(s):

José Miguel Miquelão Santos ◽

Gabriel António Amaro Monteiro ◽

Duarte Miguel de França Teixeira dos Prazeres ◽

Sofia de Oliveira Dias Duarte

Keyword(s):

Genome Editing ◽

Lactococcus Lactis ◽

Plasmid Dna ◽

Genome Engineering ◽

Gene Deletion ◽

Deletion Mutants ◽

Cell Factories ◽

Yield And Quality ◽

Delivery Vehicles

Abstract Lactococcus lactis strains are promising cell factories and delivery vehicles of plasmid DNA and recombinant protein for therapeutic applications. However, the limited yields of recombinant molecules obtained with these bacteria limits their wide applicability. Genome engineering of this host may solve the problem. However, the current genome editing toolbox available for L. lactis is either too laborious or incapable of large edits, limiting the scope of strain editing experiments. In this work, the basis for a one-plasmid CRISPR-Cas9 based genome editing plasmid was developed and tested. The new plasmid (pTCas9dO) adapted from the pKCcas9dO plasmid was used to delete 657 bp of the lactococcal nuclease nth of L. lactis subsp. lactis LMG19460, with the aim of improving yield and quality of plasmid DNA replicated in this strain. Although deletion mutants were successfully generated, plasmid curing was unsuccessful. Thus, further modifications are required before the plasmid is truly applicable for genome editing experiments. Unexpectedly, the generated deletion mutants generated a roughly 40% decrease in plasmid yield alongside with a decrease in the quality of produced pDNA.

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Modeling Non-Alcoholic Fatty Liver Disease (NAFLD) Using “Good-Fit” Genome-Editing Tools

Cells ◽

10.3390/cells9122572 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2572

Author(s):

Uijin Kim ◽

Nahyun Kim ◽

Ha Youn Shin

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Genome Editing ◽

Fatty Liver Disease ◽

Genome Engineering ◽

In Vivo Models ◽

Alcoholic Fatty Liver ◽

Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, metabolic syndrome, as well as specific genetic polymorphisms. Severe NAFLD cases can further progress to cirrhosis, hepatocellular carcinoma (HCC), or cardiovascular complications. Here, we describe the pathophysiological features and critical genetic variants associated with NAFLD. Recent advances in genome-engineering technology have provided a new opportunity to generate in vitro and in vivo models that reflect the genetic abnormalities of NAFLD. We review the currently developed NAFLD models generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. We further discuss unique features of CRISPR/Cas9 and Cas9 variants, including base editors and prime editor, that are useful for replicating genetic features specific to NAFLD. We also compare advantages and limitations of currently available methods for delivering genome-editing tools necessary for optimal genome editing. This review should provide helpful guidance for selecting “good fit” genome-editing tools and appropriate gene-delivery methods for the successful development of NAFLD models and clinical therapeutics.

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GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells

The Journal of Cell Biology ◽

10.1083/jcb.201506014 ◽

2015 ◽

Vol 211 (3) ◽

pp. 669-682 ◽

Cited By ~ 54

Author(s):

Xia Gao ◽

Aman S. Bali ◽

Scott H. Randell ◽

Brigid L.M. Hogan

Keyword(s):

Transcription Factor ◽

Genome Editing ◽

Airway Epithelium ◽

Gene Deletion ◽

Target Genes ◽

Secretory Cells ◽

Basal Cells ◽

Membrane Adhesion ◽

Regulatory Sites

Pseudostratified airway epithelium of the lung is composed of polarized ciliated and secretory cells maintained by basal stem/progenitor cells. An important question is how lineage choice and differentiation are coordinated with apical–basal polarity and epithelial morphogenesis. Our previous studies indicated a key integrative role for the transcription factor Grainyhead-like 2 (Grhl2). In this study, we present further evidence for this model using conditional gene deletion during the regeneration of airway epithelium and clonal organoid culture. We also use CRISPR/Cas9 genome editing in primary human basal cells differentiating into organoids and mucociliary epithelium in vitro. Loss of Grhl2 inhibits organoid morphogenesis and the differentiation of ciliated cells and reduces the expression of both notch and ciliogenesis genes (Mcidas, Rfx2, and Myb) with distinct Grhl2 regulatory sites. The genome editing of other putative target genes reveals roles for zinc finger transcription factor Znf750 and small membrane adhesion glycoprotein in promoting ciliogenesis and barrier function as part of a network of genes coordinately regulated by Grhl2.

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