Naturally Occurring Off-Switches for CRISPR-Cas9

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcı́a, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J.; Maxwell, Karen L.; Davidson, Alan R.

doi:10.1016/j.cell.2016.11.017

Cited by 366 publications

(441 citation statements)

References 66 publications

(109 reference statements)

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“…In order to reduce unspecific genomic changes by the nickase, CRISPR-Cas9 inhibitory proteins could be expressed to inhibit the activity of Cas9 after genomic editing (35,36). The prophage-encoded inhibitor proteins AcrIIA2 and AcrIIA4, which allow phages to evade the bacterial host's CRISPR/Cas immune system, were found to inhibit Cas9-based targeting in their native host Listeria monocytogenes, as well as Cas9 of Streptococcus pyogenes in bacteria and human cells.…”

Section: Discussionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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Section: Discussionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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“…3a), since the Aca proteins need not necessarily engage any specific CRISPR-Cas system components directly. An aca2 ortholog (with a small, novel ORF upstream of it) was indeed identified in a putative MGE in a strain of Brackiella oedipodis that harbored a Type II-C CRISPR- cas locus but no Type I system [30]. The adjacent candidate acr gene had an ortholog in a strain of Neisseria meningitidis that was adjacent to a distinct candidate aca gene ( aca3 ).…”

Section: Type II Anti-crisprsmentioning

confidence: 99%

“…3a, several candidate Type II-C anti-CRISPRs were compiled, three of which were in a species readily amenable to functional analysis. Tests of native CRISPR interference in N. meningitidis 8013 validated all three meningococcal candidates (AcrIIC1 Nme , AcrIIC2 Nme , and AcrIIC3 Nme ) as bona fide Type II-C anti-CRISPRs [30]. Intriguingly, the B. oedipodis candidate (AcrIIC1 Boe ) was also found to prevent interference by the native NmeCas9 (~47% identical to B. oedipidis Cas9), indicating cross-species inhibition and suggesting that some type II anti-CRISPRs could be broad-spectrum Cas9 inhibitors.…”

Section: Type II Anti-crisprsmentioning

confidence: 99%

Inhibition of CRISPR-Cas systems by mobile genetic elements

Sontheimer

Davidson

2017

Current Opinion in Microbiology

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Clustered, regularly interspaced, short, palindromic repeats (CRISPR) loci, together with their CRISPR-associated (Cas) proteins, provide bacteria and archaea with adaptive immunity against invasion by bacteriophages, plasmids, and other mobile genetic elements. These host defenses impart selective pressure on phages and mobile elements to evolve countermeasures against CRISPR immunity. As a consequence of this pressure, phages and mobile elements have evolved “anti-CRISPR” proteins that function as direct inhibitors of diverse CRISPR-Cas effector complexes. Some of these CRISPR-Cas complexes can be deployed as genome engineering platforms, and anti-CRISPRs could therefore be useful in exerting spatial, temporal, or conditional control over genome editing and related applications. Here we describe the discovery of anti-CRISPRs, the range of CRISPR-Cas systems that they inhibit, their mechanisms of action, and their potential utility in biotechnology.

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“…Phages can evolve or acquire counter-systems to circumvent bacterial immunity [79]. For example, some phages have produced their own methyltransferases to defend against bacterial restriction-modification systems [80], whereas other phages encode for anti-CRISPR-Cas systems [79,81].…”

Section: Phage Resistancementioning

confidence: 99%

Phage therapy: awakening a sleeping giant

Roach

Debarbieux

2017

Emerging Topics in Life Sciences

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Correspondence: Laurent Debarbieux (laurent.debarbieux@pasteur.fr)For a century, bacterial viruses called bacteriophages have been exploited as natural antibacterial agents. However, their medicinal potential has not yet been exploited due to readily available and effective antibiotics. After years of extensive use, both properly and improperly, antibiotic-resistant bacteria are becoming more prominent and represent a worldwide public health threat. Most importantly, new antibiotics are not progressing at the same rate as the emergence of resistance. The therapeutic modality of bacteriophages, called phage therapy, offers a clinical option to combat bacteria associated with diseases. Here, we discuss traditional phage therapy approaches, as well as how synthetic biology has allowed for the creation of designer phages for new clinical applications. To implement these technologies, several key aspects and challenges still need to be addressed, such as narrow spectrum, safety, and bacterial resistance. We will summarize our current understanding of how phage treatment elicits mammalian host immune responses, as well bacterial phage resistance development, and the potential impact each will have on phage therapy effectiveness. We conclude by discussing the need for a paradigm shift on how phage therapy strategies are developed.

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Naturally Occurring Off-Switches for CRISPR-Cas9

Cited by 366 publications

References 66 publications

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Inhibition of CRISPR-Cas systems by mobile genetic elements

Phage therapy: awakening a sleeping giant

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