scholarly journals Improved sgRNA design in bacteria via genome-wide activity profiling

2018 ◽  
Author(s):  
Jiahui Guo ◽  
Tianmin Wang ◽  
Changge Guan ◽  
Bing Liu ◽  
Cheng Luo ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Large Scale ◽  
Genome Engineering ◽  
Prediction Models ◽  
Prokaryotic Genome ◽  
Model Organism ◽  
Bacterial Cells ◽  
Highly Active ◽  
Promising Tool ◽  
A Genome

AbstractCRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (~70,000 sgRNAs) with Cas9 or its specificity-improved mutant in E. coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified ‘resistant’ genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate the design of CRISPR/Cas9-based genome engineering or screenings in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

scholarly journals Prediction of activity of CRISPR-Cpf1 guide RNA via in vivo high-throughput screening

2021 ◽  
Author(s):  
gancheng wang ◽  
dan zhu ◽  
juan li ◽  
junyi wang ◽  
jianzhong xi
Keyword(s):  
Dna Cleavage ◽  
High Throughput Screening ◽  
Large Scale ◽  
Genome Engineering ◽  
Limited Information ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Guide Rnas ◽  
Promising Tool

Background: CRISPR-cpf1 is a single RNA-guided endonuclease system, becoming a promising tool in both prokaryotic and eukaryotic genome engineering. The editing efficiency of Cpf1 based engineering still requires improvements. However, limited information regarding the relationship between guide RNA sequence and on-target activity is available. To address these challenges, we developed a screening platform based on the association of Acidaminococcus sp. Cpf1(AsCpf1) DNA cleavage with cellular lethality. Major results: In total, we measured the activities of 12,544 guide RNAs, and observed a substantial variation of the editing efficiency depending on the design of the sequence. Based on this large-scale dataset, we designed and implemented a comprehensive computational model to predict activities of guide RNAs. Through comparison using simulated and experimental data, our approach outperformed existing algorithms, enabling selection of efficient guide RNAs. Conclusions: We refine on-target design rules and isolate the important sequence features that contribute to DNA cleavage, that is, AH dimers at position1-8 of protospacer promoting Cas12a activity while TK, GB dimer playing an inhibitory role. We validate guide RNA affinities designed by our optimized rules in both E.coli and 293T cells.

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

2019 ◽  
Vol 18 (1) ◽  
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.

scholarly journals Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1828
Author(s):  
Francesco Citiulo ◽  
Cristina Crosatti ◽  
Luigi Cattivelli ◽  
Chiara Biselli
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Fast Response ◽  
Scale Production ◽  
Bacterial Cells ◽  
Regulatory Requirements ◽  
Emergency Situations ◽  
Large Scale Production ◽  
A Genome ◽  
Transformation Systems

The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles - VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.

scholarly journals Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA

2020 ◽  
Vol 48 (10) ◽  
pp. 5527-5539 ◽  
Author(s):  
Zongliang Gao ◽  
Minghui Fan ◽  
Atze T Das ◽  
Elena Herrera-Carrillo ◽  
Ben Berkhout
Keyword(s):  
Cell Culture ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Viral Escape ◽  
Sequence Profile ◽  
Guide Rna ◽  
Complete Inactivation ◽  
Promising Tool ◽  
Immunodeficiency Virus ◽  
Increased Activity

Abstract The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.

Construction of a minimal genome as a chassis for synthetic biology

2016 ◽  
Vol 60 (4) ◽  
pp. 337-346 ◽  
Author(s):  
Bong Hyun Sung ◽  
Donghui Choe ◽  
Sun Chang Kim ◽  
Byung-Kwan Cho
Keyword(s):  
Synthetic Biology ◽  
Genome Stability ◽  
Large Scale ◽  
Genome Engineering ◽  
Essential Gene ◽  
Industrial Applications ◽  
Microbial Genomes ◽  
Gene Set ◽  
Minimal Genome ◽  
A Genome

Microbial diversity and complexity pose challenges in understanding the voluminous genetic information produced from whole-genome sequences, bioinformatics and high-throughput ‘-omics’ research. These challenges can be overcome by a core blueprint of a genome drawn with a minimal gene set, which is essential for life. Systems biology and large-scale gene inactivation studies have estimated the number of essential genes to be ∼300–500 in many microbial genomes. On the basis of the essential gene set information, minimal-genome strains have been generated using sophisticated genome engineering techniques, such as genome reduction and chemical genome synthesis. Current size-reduced genomes are not perfect minimal genomes, but chemically synthesized genomes have just been constructed. Some minimal genomes provide various desirable functions for bioindustry, such as improved genome stability, increased transformation efficacy and improved production of biomaterials. The minimal genome as a chassis genome for synthetic biology can be used to construct custom-designed genomes for various practical and industrial applications.

scholarly journals Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans

2020 ◽  
Author(s):  
Troy A. McDiarmid ◽  
Vinci Au ◽  
Donald G. Moerman ◽  
Catharine H. Rankin
Keyword(s):  
Caenorhabditis Elegans ◽  
Machine Vision ◽  
Negative Selection ◽  
Large Scale ◽  
Genome Engineering ◽  
Model Organism ◽  
Model Organisms ◽  
Behavioral Phenotypes ◽  
Functional Studies ◽  
Marker Selection

AbstractImproved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome edited integrants, often circumventing the need for visual screening to identify genome edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome edited animals without the need for screening or scoring by eye.Author summaryThe ability to directly manipulate the genome and observe the resulting effects on the traits of an organism is a powerful approach to investigate gene function. CRISPR-based approaches to genome engineering have revolutionized such functional studies across model organisms but still face major challenges that limit the scope and complexity of projects that can be achieved in practice. Automating genome engineering and phenotyping would enable large-scale investigations of genome function in animals. Here, we describe the adaptation of peel-1 negative selection to an optimized dual-marker selection cassette CRISPR-Cas9 genome engineering method in C. elegans and combine it with automated machine vision phenotyping to achieve functional studies without the need for screening or scoring by eye. To demonstrate the applicability of the approach, we generated novel deletion alleles in two understudied genes, pbs-1 and K04F10.3, and used machine vision to characterize their phenotypic profiles, revealing homozygous lethal and heterozygous behavioral phenotypes. Our results open the door to systematic investigations of genome function in this model organism.

scholarly journals Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies

2019 ◽  
Author(s):  
Jonathan Zirin ◽  
Yanhui Hu ◽  
Luping Liu ◽  
Donghui Yang-Zhou ◽  
Ryan Colbeth ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Gene Activation ◽  
Somatic Tissue ◽  
Transcription Start ◽  
Synergistic Activation ◽  
A Genome ◽  
Or Genes ◽  
Genome Scale ◽  
Transgenic Drosophila

ABSTRACTThe Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

scholarly journals Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 755-767 ◽  
Author(s):  
Jonathan Zirin ◽  
Yanhui Hu ◽  
Luping Liu ◽  
Donghui Yang-Zhou ◽  
Ryan Colbeth ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Gene Activation ◽  
Somatic Tissue ◽  
Guide Rnas ◽  
Synergistic Activation ◽  
A Genome ◽  
Or Genes ◽  
Genome Scale ◽  
Transgenic Drosophila

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

scholarly journals CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

2021 ◽  
Author(s):  
Jun Huang ◽  
David Rowe ◽  
Wei Zhang ◽  
Tyler Suelter ◽  
Barbara Valent ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Cleavage ◽  
Large Scale ◽  
Genome Engineering ◽  
Natural Populations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Long Read ◽  
Repair Pathways

AbstractCRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

Bioactivity Prediction Based on Matched Molecular Pair and Matched Molecular Series Methods

2020 ◽  
Vol 26 (33) ◽  
pp. 4195-4205
Author(s):  
Xiaoyu Ding ◽  
Chen Cui ◽  
Dingyan Wang ◽  
Jihui Zhao ◽  
Mingyue Zheng ◽  
...  
Keyword(s):  
Prediction Model ◽  
Large Scale ◽  
Prediction Models ◽  
Predictive Accuracy ◽  
Lead Optimization ◽  
Consensus Method ◽  
Molecular Pair ◽  
Bioactivity Prediction ◽  
Compound Synthesis ◽  
Consensus Modeling

Background: Enhancing a compound’s biological activity is the central task for lead optimization in small molecules drug discovery. However, it is laborious to perform many iterative rounds of compound synthesis and bioactivity tests. To address the issue, it is highly demanding to develop high quality in silico bioactivity prediction approaches, to prioritize such more active compound derivatives and reduce the trial-and-error process. Methods: Two kinds of bioactivity prediction models based on a large-scale structure-activity relationship (SAR) database were constructed. The first one is based on the similarity of substituents and realized by matched molecular pair analysis, including SA, SA_BR, SR, and SR_BR. The second one is based on SAR transferability and realized by matched molecular series analysis, including Single MMS pair, Full MMS series, and Multi single MMS pairs. Moreover, we also defined the application domain of models by using the distance-based threshold. Results: Among seven individual models, Multi single MMS pairs bioactivity prediction model showed the best performance (R2 = 0.828, MAE = 0.406, RMSE = 0.591), and the baseline model (SA) produced the most lower prediction accuracy (R2 = 0.798, MAE = 0.446, RMSE = 0.637). The predictive accuracy could further be improved by consensus modeling (R2 = 0.842, MAE = 0.397 and RMSE = 0.563). Conclusion: An accurate prediction model for bioactivity was built with a consensus method, which was superior to all individual models. Our model should be a valuable tool for lead optimization.

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