Overview of CRISPR–Cas9 Biology

Ratner, Hannah K.; Sampson, Timothy R.; Weiss, David S.

doi:10.1101/pdb.top088849

Cited by 19 publications

(15 citation statements)

References 165 publications

(338 reference statements)

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“…The RecA-like protein fusion successfully imported a restriction enzyme but failed to import Cas9 [ 26 ]. Therefore, we tested the ability of GFPmut1 to import three proteins from different CRISPR-cas systems: Cas3, Cas9, and Cas13 [ 27 – 31 ]. When fused to sfGFP, all three localized in the cytoplasm, indicating that all three of these proteins are normally excluded from the nucleus ( Fig 5A, 5C and 5E ).…”

Section: Resultsmentioning

confidence: 99%

Selective transport of fluorescent proteins into the phage nucleus

et al. 2021

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Upon infection of Pseudomonas cells, jumbo phages 201Φ2–1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2–1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

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Section: Resultsmentioning

confidence: 99%

Selective transport of fluorescent proteins into the phage nucleus

et al. 2021

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“…Spacers are transcribed into small RNAs that guide the direct cleavage of viral DNA by Cas nuclease proteins. The immunized population not only acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny [ 42 , 43 ]. In this study, we observed that E. faecalis genomes contain a significantly higher number of recombination hotspots than E. faecium genomes do.…”

Section: Discussionmentioning

confidence: 99%

Extensive Comparative Genomic Analysis of Enterococcus faecalis and Enterococcus faecium Reveals a Direct Association between the Absence of CRISPR–Cas Systems, the Presence of Anti-Endonuclease (ardA) and the Acquisition of Vancomycin Resistance in E. faecium

et al. 2021

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Here, we performed a comparative genomic analysis of all available genomes of E. faecalis (n = 1591) and E. faecium (n = 1981) and investigated the association between the presence or absence of CRISPR-Cas systems, endonuclease/anti-endonuclease systems and the acquisition of antimicrobial resistance, especially vancomycin resistance genes. Most of the analysed Enterococci were isolated from humans and less than 14% of them were from foods and animals. We analysed and detected CRISPR–Cas systems in 75.36% of E. faecalis genomes and only 4.89% of E. faecium genomes with a significant difference (p-value < 10−5). We found a negative correlation between the number of CRISPR–Cas systems and genome size (r = −0.397, p-value < 10−5) and a positive correlation between the genome %GC content and the number of CRISPR–Cas systems (r = 0.215, p-value < 10−5). Our findings showed that the presence of the anti-endonuclease ardA gene may explain the decrease in the number of CRISPR–Cas systems in E. faecium, known to deactivate the endonucleases’ protective activities and enable the E. faecium genome to be versatile in acquiring mobile genetic elements, including carriers of antimicrobial resistance genes, especially vanB. Most importantly, we observed that there was a direct association between the absence of CRISPR–Cas, the presence of the anti-CRISPR ardA gene and the acquisition of vancomycin resistance genes.

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“…The RecA-like protein fusion successfully imported a restriction enzyme but failed to import Cas9 (26). Therefore, we tested the ability of GFPmut1 to import three proteins from different CRISPR-cas systems: Cas3, Cas9, and Cas13 (27–31). When fused to sfGFP, all three localized in the cytoplasm, indicating that all three of these proteins are normally excluded from the nucleus (Fig 5A, C, E).…”

Section: Resultsmentioning

confidence: 99%

Selective transport of fluorescent proteins into the phage nucleus

Nguyen

Sugie

Khanna

et al. 2020

Preprint

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Upon infection of Pseudomonas cells, jumbo phages 201Φ2-1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

show abstract

Overview of CRISPR–Cas9 Biology

Cited by 19 publications

References 165 publications

Selective transport of fluorescent proteins into the phage nucleus

Selective transport of fluorescent proteins into the phage nucleus

Extensive Comparative Genomic Analysis of Enterococcus faecalis and Enterococcus faecium Reveals a Direct Association between the Absence of CRISPR–Cas Systems, the Presence of Anti-Endonuclease (ardA) and the Acquisition of Vancomycin Resistance in E. faecium

Selective transport of fluorescent proteins into the phage nucleus

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