Shutting down RNA-targeting CRISPR

doi:10.1126/science.abc8243

GSTDTAP > 气候变化

DOI	10.1126/science.abc8243
	Shutting down RNA-targeting CRISPR
	Rodolphe Barrangou; Erik J. Sontheimer
	2020-07-03
发表期刊	Science
出版年	2020
英文摘要	Explorations of the evolutionary arms race between bacteria and bacteriophages (viruses that infect bacteria) have unearthed a variety of defense mechanisms that include CRISPR-Cas (CRISPR-associated nuclease) adaptive immune systems ([ 1 ][1]). Understanding the mechanisms of CRISPR-mediated immunity, involving DNA-encoded, RNA-guided, sequence-specific targeting of invasive nucleic acids ([ 2 ][2], [ 3 ][3]), has spawned powerful genome engineering platforms based on diverse Cas effectors. Subsequent studies have also revealed anti-CRISPR proteins ([ 4 ][4]) that have proven valuable as control switches for Cas molecular machines. CRISPR-Cas immune systems encompass diverse families including DNA-targeting effectors such as Cascade-Cas3, Cas9, and Cas12 as well as the recently characterized RNA-targeting Cas13 ribonuclease (RNase) ([ 5 ][5]). The evolving immune arsenal in bacteria has been matched by diverse anti-CRISPRs that enable viruses to escape Cas nuclease targeting. On page 54 of this issue, Meeske et al. ([ 6 ][6]) report an anti-CRISPR mechanism that inhibits Cas13a RNase activity, with potential utility as a CRISPR effector control switch. The Cas13 RNase is the signature protein for type VI CRISPR-Cas systems ([ 5 ][5]), which use their CRISPR RNA (crRNA) guides to target viral RNA sequences and trigger both the specific degradation of target phage RNA and the nonspecific destruction of host transcripts ([ 7 ][7]), leading to host dormancy and prevention of virus proliferation ([ 8 ][8]). Meeske et al. identified and characterized a type VI anti-CRISPR protein, AcrVIA1Lse, encoded by the genome of the ϕLS46 listeriophage (see the figure). This anti-CRISPR binds to Listeria seeligeri Cas13a (LsCas13a) and prevents target RNA access and conformational activation of the RNase. AcrVIA1Lse comprises 232 amino acids, which is notably larger than the typically hypervariable, <150–amino acid anti-CRISPRs previously characterized. The additional residues augment the RNase domain with a DNA-binding motif that contributes to anti-CRISPR transcriptional regulation. This AcrVIA1Lse protein was able to completely thwart CRISPR-Cas13 immunity against otherwise susceptible listeriophage. Meeske et al. investigated the interplay between AcrVIA1Lse and the Cas13a:crRNA complex and showed dose-dependent inhibition of RNase activity through formation of an AcrVIA1Lse:Cas13a:crRNA complex that prevents target RNA binding. Critically, both cis phage-targeting and trans host dormancy–inducing activities are abolished by AcrVIA1Lse, enhancing not only the capacity of the listeriophage to replicate but also the ability of the host L. seeligeri to continue metabolically. This interaction was dissected structurally, and the authors used cryo–electron microscopy to establish that LsCas13a adopts a bilobed architecture encompassing a recognition domain and an RNase domain, directed by a 51-nucleotide crRNA with the target-complementary portion configured to pair with viral RNA. AcrVIA1Lse interacts with both LsCas13a and the crRNA to prevent target RNA binding and RNase activation. Future studies will undoubtedly define AcrVIA1Lse inhibitory specificity across Cas13 homologs, as well as the impact of type VI anti-CRISPR viral resistance on bacterial biology and host-virus dynamics. Several studies have deciphered how anti-CRISPRs can inhibit Cas effectors by blocking target nucleic acid loading and preventing Cas nuclease activity ([ 4 ][4]), but the primary focus had been on DNA-targeting Cascade-Cas3, Cas9, and Cas12. These studies revealed that physiological anti-CRISPR activity often only partially inhibits CRISPR-Cas, providing limited immune escape ([ 9 ][9], [ 10 ][10]). This appears to reflect a race between anti-CRISPR expression in phages and host immune defense by the CRISPR effector complexes. Viral infections can fail because anti-CRISPR expression does not reach inhibitory thresholds in time to overcome CRISPR immunity, but repeated failed infections can yield gradually increasing anti-CRISPR amounts that immunosuppress the host and allow later infections to succeed. In the case of type VI CRISPR immunity, however, the DNA genome of the invasive phage is not specifically cleaved; instead, viral and host transcripts are the targets for Cas13-mediated degradation, and virus propagation is suppressed primarily by the induction of bacterial host cell dormancy ([ 8 ][8]). This fundamental distinction reduces the time pressure on anti-CRISPR–mediated inhibition of Cas13 because the phage genome is not specifically cleared and dormancy can often be reversed. ![Figure][11] Inhibiting CRISPR-associated ribonuclease activity Listeriophage LS46 encodes the anti-CRISPR protein AcrVIA1Lse. The host, Listeria seeligeri , encodes a type VI CRISPR-Cas13a (CRISPR-associated 13a) immune system. Cas13a is a ribonuclease that, through complementarity between CRISPR RNA (crRNA) and target sequences in listeriophage messenger RNA (mRNA), cleaves and thus inactivates phage mRNA in cis and host mRNA in trans. AcrVIA1Lse can bind and inhibit the Cas13a:crRNA complex. GRAPHIC: A. KITTERMAN/ SCIENCE A single dose of AcrVIA1Lse could completely abrogate LsCas13 activity, even in unfavorable conditions such as very low viral multiplicity of infection, targeting by multiple crRNAs, or immunity activation by early phage transcripts. Thus, mechanistically establishing how these different types of CRISPR systems function is a critical step toward understanding and repurposing these molecular machines and ultimately manipulating, engineering, and controlling Cas13-based tools. Besides the continued rise of DNA-targeting CRISPR effectors such as Cas9 and Cas12 for genome editing, several recent studies have illustrated how valuable RNA-targeting Cas13 effectors can be, including for detection of RNA viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19) ([ 11 ][12]). There is also much potential for Cas13 as an RNA-targeting and RNA-editing platform (for example, to correct disease-causing mutations in cellular RNAs) ([ 12 ][13], [ 13 ][14]), but this depends on whether its activity can be controlled. The study of Meeske et al. provides a means to reduce or eliminate Cas13 activity, opening new avenues for the use of CRISPR to control and shape the transcriptomes of organisms and viruses. Indeed, type VI anti-CRISPRs can readily control Cas13a RNA targeting and dCas13a RNA editing in human cells ([ 14 ][15]). However, more studies are needed to harness this tool and establish how broadly exploitable such Cas13-based technologies (and their off switches) could be. 1. [↵][16]1. R. Barrangou et al ., Science 315, 1709 (2007). [OpenUrl][17][Abstract/FREE Full Text][18] 2. [↵][19]1. S. J. Brouns et al ., Science 321, 960 (2008). [OpenUrl][20][Abstract/FREE Full Text][21] 3. [↵][22]1. L. A. Marraffini, 2. E. J. Sontheimer , Science 322, 1843 (2008). [OpenUrl][23][Abstract/FREE Full Text][24] 4. [↵][25]1. A. R. Davidson et al ., Annu. Rev. Biochem. 10.1146/annurev-biochem-011420-111224 (2020). 5. [↵][26]1. K. S. Makarova et al ., Nat. Rev. Microbiol. 18, 67 (2020). [OpenUrl][27][CrossRef][28][PubMed][29] 6. [↵][30]1. A. J. Meeske et al ., Science 369, 54 (2020). [OpenUrl][31][Abstract/FREE Full Text][32] 7. [↵][33]1. O. O. Abudayyeh et al ., Science 353, aaf5573 (2016). [OpenUrl][34][Abstract/FREE Full Text][35] 8. [↵][36]1. A. J. Meeske et al ., Nature 570, 241 (2019). [OpenUrl][37] 9. [↵][38]1. A. L. Borges et al ., Cell 174, 917 (2018). [OpenUrl][39] 10. [↵][40]1. M. Landsberger et al ., Cell 174, 908 (2018). [OpenUrl][41] 11. [↵][42]1. C. M. Ackerman et al ., Nature 582, 277 (2020). [OpenUrl][43] 12. [↵][44]1. O. O. Abudayyeh et al ., Nature 550, 280 (2017). [OpenUrl][45][CrossRef][46][PubMed][47] 13. [↵][48]1. D. B. T. Cox et al ., Science 358, 1019 (2017). [OpenUrl][49][Abstract/FREE Full Text][50] 14. [↵][51]1. P. Lin et al ., Mol. Cell 78, 850 (2020). [OpenUrl][52] Acknowledgments: R.B. and E.J.S. are co-founders and advisers of Intellia Therapeutics, a company involved in the development of CRISPR-based therapies. 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领域	气候变化 ; 资源环境
URL	查看原文
引用统计
文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/281857
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Rodolphe Barrangou,Erik J. Sontheimer. Shutting down RNA-targeting CRISPR[J]. Science,2020.
APA	Rodolphe Barrangou,&Erik J. Sontheimer.(2020).Shutting down RNA-targeting CRISPR.Science.
MLA	Rodolphe Barrangou,et al."Shutting down RNA-targeting CRISPR".Science (2020).