ge-CRISPR - An integrated pipeline for the prediction and analysis of sgRNAs genome editing efficiency for CRISPR/Cas system

Kaur, Karambir; Gupta, Amit; Rajput, Abhishek; Kumar, Manoj

doi:10.1038/srep30870

Cited by 43 publications

(24 citation statements)

References 55 publications

(86 reference statements)

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“…Whether the activity of these sgRNAs could be predicted by the publicly available sgRNA design tools was then determined. All 87 sgRNAs were scored using sgRNA Scorer 2.0 (29) and ge-CRISPR (30), which were trained from eukaryotic data sets. The predicted score then was compared with the observed activity ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Shen

Zhou

Chen

et al. 2018

J Virol

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is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from and a single guide RNA (sgRNA) were used to modify a virulent bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy. In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.

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Section: Resultsmentioning

confidence: 99%

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Shen

Zhou

Chen

et al. 2018

J Virol

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“…Upon inspecting the structure of the SpCas9 RNP poised to cleave a DNA target (Jiang et 340 al., 2016), we concluded that sequences adjacent to and upstream of the PAM sequence (5'-NGG-3'), which have been reported to bias SpCas9 activity in vivo (Doench et al, 2014;Wu et al, 2014;Xu et al, 2015;Moreno-Mateos et al, 2015;Horlbeck et al, 2016;Liu et al, 2016;Kaur et al, 2016;Gandhi et al, 2017), likely influence SpCas9 recognition by stabilizing the conformation of the DNA target for cleavage. Therefore, we accounted for biases in SpCas9 345 recognition by choosing gRNAs sequences that conformed, as much as possible, to the motif 5'-HNNGRSGGH-3', in which the PAM is underlined, N stands for any base, R stands for purine, S stands for G or C, and H stands for any base except G. This motif was first used to search for suitable targets (Peng and Tarleton, 2015) in cDNA sequences.…”

Section: Design and Preparation Of Grnasmentioning

confidence: 87%

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

Booth

King

2020

Preprint

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In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene 25 function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This 30 work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

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“…Genus Brachybacterium are widely distributed in various environments. From marine environments, B. aquaticum has been successfully isolated from seawater (Kaur et al, 2016). Zhang et al, (2007) reported that Brachybacterium zhongshanense sp.…”

Section: Molecular Identification Of Bacterial Strain Sj04mentioning

confidence: 99%

CHARACTERIZATION OF CARTENOID PIGMENTS FROM Sargassum polycystum AND ITS ASSOCIATED BACTERIA

Kusmita¹,

Nugraheni²,

Nuryadi³

2019

Pak J. Biotech

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Several bacteria are capable to produce various carotenoid pigments, and they may become potential bioresources for pigment production. Bacteria produce pigments for various reasons and it plays an important role. They may help to protect their host from high exposure of UV-light. However, the ability of marine bacteria to produce natural pigments has been less studied. In this study, we tried to isolate and characterize marine pigment-producing bacteria of brown seaweed macroalgae, Sargassum polycystum from Teluk Awur, Jepara. Out of seven bacterial isolates, only one bacterium, SJ04, positively contains pigments. Pigments analysis using HPLC method showed that pigment composition was slightly different between host and associated bacterium. Pheophorbide a, Fucoxanthin, and β-caroten were detected from S. polycystum as host, whereas Pheophorbide a, and Neoxanthin were detected from the associated bacteria (SJ04). For bacterium identification, molecular genetic approach based on 16S rRNA gene sequence showed that strain SJ04 was genetically closely related to Brachybacterium zhongshanense with 97.97% homology. The opportunity to realize a safe and environmentally friendly with low price , as well as opportunities to find new sources of pigments from bacteria.

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ge-CRISPR - An integrated pipeline for the prediction and analysis of sgRNAs genome editing efficiency for CRISPR/Cas system

Cited by 43 publications

References 55 publications

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

CHARACTERIZATION OF CARTENOID PIGMENTS FROM Sargassum polycystum AND ITS ASSOCIATED BACTERIA

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