Inhibition of CRISPR-Cas systems by mobile genetic elements

Sontheimer, Erik J.; Davidson, Alan R.

doi:10.1016/j.mib.2017.06.003

Cited by 34 publications

(33 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To counteract CRISPR-Cas immunity, phages employ inhibitory proteins to inactivate CRISPR-Cas function in a sequenceindependent manner (Bondy-Denomy et al, 2013;Sontheimer and Davidson, 2017). To date, >40 diverse anti-CRISPRs have been identified in phages, prophages, and mobile genetic elements (Borges et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

et al. 2019

View full text Add to dashboard Cite

show abstract

Section: Introductionmentioning

confidence: 99%

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

et al. 2019

View full text Add to dashboard Cite

show abstract

“…However, just one of the 130 18 contigs from this library was found to be phage-associated (table S4) (table S3). Many contigs from these libraries are phage-associated (30%; table S4, 141 figure 2A) and many encode genes of unknown function (figure S4); these are both hallmarks of known 142 acrs (Borges et al, 2017;Pawluk et al, 2017;Sontheimer and Davidson, 2017). Additionally, we 143 observed that Cas9 homologs are found in 15 of the 18 bacterial genera identified in these selections 144 (83%, table S4), a significant enrichment over the 12% of bacterial genera that harbor Cas9 (567/4822) 145 from a representative set of ~24,000 bacterial genomes (Mendler et al, 2018) (p=4x10 -21 , χ² test).…”

Section: Introduction 14mentioning

confidence: 99%

“…These clever strategies have revealed many 49 candidate acrs. But they have also highlighted the difficulty of finding new acr genes based on homology, 50 since acrs share little sequence conservation (Sontheimer and Davidson, 2017). As a result, most acrs 51 almost certainly lie unrecognized among the many genes of unknown function in phages, plasmids, and 52 other MGEs (Hatfull, 2015).…”

mentioning

confidence: 99%

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Forsberg

Bhatt

Schmidtke

et al. 2019

Preprint

View full text Add to dashboard Cite

1 CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, 2 which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are 3 difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. 4To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates 5 acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten 6 DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 7 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage 8 and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple 9 bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes 10 SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study 11 highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse 12 microbiomes. 13 4 an Acr protein Watters et al., 2018). These clever strategies have revealed many 49 candidate acrs. But they have also highlighted the difficulty of finding new acr genes based on homology, 50 since acrs share little sequence conservation (Sontheimer and Davidson, 2017). As a result, most acrs 51 almost certainly lie unrecognized among the many genes of unknown function in phages, plasmids, and 52 other MGEs (Hatfull, 2015). 53 54To overcome the challenges associated with anti-CRISPR discovery, we devised a functional 55 metagenomic selection that identifies acr genes from any cloned DNA, based on their ability to protect a 56 plasmid from CRISPR-Cas-mediated destruction. Because functional metagenomics selects for a 57 function of interest from large clone libraries (Handelsman, 2004), it is well-suited to identify individual 58 genes like acrs that have strong fitness impacts (Iqbal et al., 2014;Forsberg et al., 2015; Forsberg et al., 59 2016; Genee et al., 2016). This approach may be particularly useful for Acr discovery because Acrs are 60 expressed from single genes and function readily in many genetic backgrounds (Pawluk et al., 2016a; 61 Rauch et al., 2017). 62 63Using this functional selection, we find that many unrelated metagenomic clones from human oral 64 and gut microbiomes protect against Streptococcus pyogenes Cas9 (SpyCas9), the variant used most 65 commonly for gene editing applications (Knott and Doudna, 2018). We identify a broadly distributed but 66 previously undescribed Acr from the most potent Cas9-antagonizing clone in our libraries. This Acr, 67 named AcrIIA11, binds both SpyCas9 and double-stranded DNA (dsDNA) and exhibits a novel mode of 68 SpyCas9 antagonism, protecting both plasmids and phages from immune restriction. Thus, our functional 69 approach reveals not only new classes of Acrs, but also new mechanisms of CRISPR-Cas antagonism. 70 71Results 72 73 A functio...

show abstract

“…In this pipeline, new acr gene candidates are identified by their proximity to predicted helix-turn-helix (HTH) transcriptional regulator genes known as anti-CRISPR associated ( aca ) genes 16 . We identified ORFs encoding uncharacterized small [~50-150 amino acids (aa)] proteins immediately upstream of aca homologues, focusing on genomic regions near putative phage- or MGE- associated sequences 3, 4, 24 . These criteria led us to focus on two putative Acr candidates: an 88 aa hypothetical protein in the genome of H. parainfluenzae strain 146_HPAR (NCBI RefSeq accession WP_049372635) and a 120aa hypothetical protein in the genome of S. muelleri strain ATCC 29453 (WP_002642161.1; Supplementary Table 1).…”

Section: Resultsmentioning

confidence: 99%

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

Lee

Mir

Edraki

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2–4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

show abstract

Inhibition of CRISPR-Cas systems by mobile genetic elements

Cited by 34 publications

References 51 publications

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

Contact Info

Product

Resources

About