Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression

Qi, Lei; Larson, Matthew H.; Gilbert, Luke A.; Doudna, Jennifer A.; Weissman, Jonathan S.; Arkin, Adam P.; Lim, Wendell A.

doi:10.1016/j.cell.2021.01.019

Cited by 113 publications

(123 citation statements)

References 0 publications

Supporting

Mentioning

117

Contrasting

Unclassified

Order By: Relevance

“…Cas-Effector Fusion Platforms CRISPR interference and activation (CRISPRi/a). In addition to its use for gene editing, Cas9 has been repurposed as an RNAguided DNA binding domain by introducing mutations in the RuvC and HNH nuclease domains (Bikard et al, 2013;Qi et al, 2013). In mammalian cells, robust gene repression was achieved by fusing a nuclease-deactivated Cas9 (dCas9) to a repressor domain like the Krü ppel-associated box (KRAB) domain and targeting it to the transcription start site (Gilbert et al, 2014;Gilbert et al, 2013) (Figure 2A).…”

Section: Human Health Applications Editing Strategies Based On Nuclease Activitymentioning

confidence: 99%

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

Wang

Zhang

Gao

2020

Cell

344

261

View full text Add to dashboard Cite

Section: Human Health Applications Editing Strategies Based On Nuclease Activitymentioning

confidence: 99%

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

Wang

Zhang

Gao

2020

Cell

344

261

View full text Add to dashboard Cite

“…A catalytically inactive version of the Cas9 protein called dead, or deactivated, Cas9 (dCas9) ( 177 ) repurposes the CRISPR-Cas9 platform for precision edited of the epigenome or gene expression machinery ( 173 – 175 , 177 ). A diverse spectrum of epigenetic effectors has been tethered to dCas9 to deliver epigenetic payloads to specific sites across the genome, giving rise to a continually expanding epigenome editing toolkit ( 173 , 178 ).…”

Section: Bioengineering Approaches To Reverse Epigenetic-mediated Immune Exhaustion and Suppressionmentioning

confidence: 99%

Reversing Post-Infectious Epigenetic-Mediated Immune Suppression

et al. 2021

View full text Add to dashboard Cite

The immune response must balance the pro-inflammatory, cell-mediated cytotoxicity with the anti-inflammatory and wound repair response. Epigenetic mechanisms mediate this balance and limit host immunity from inducing exuberant collateral damage to host tissue after severe and chronic infections. However, following treatment for these infections, including sepsis, pneumonia, hepatitis B, hepatitis C, HIV, tuberculosis (TB) or schistosomiasis, detrimental epigenetic scars persist, and result in long-lasting immune suppression. This is hypothesized to be one of the contributing mechanisms explaining why survivors of infection have increased all-cause mortality and increased rates of unrelated secondary infections. The mechanisms that induce epigenetic-mediated immune suppression have been demonstrated in-vitro and in animal models. Modulation of the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR), nuclear factor of activated T cells (NFAT) or nuclear receptor (NR4A) pathways is able to block or reverse the development of detrimental epigenetic scars. Similarly, drugs that directly modify epigenetic enzymes, such as those that inhibit histone deacetylases (HDAC) inhibitors, DNA hypomethylating agents or modifiers of the Nucleosome Remodeling and DNA methylation (NuRD) complex or Polycomb Repressive Complex (PRC) have demonstrated capacity to restore host immunity in the setting of cancer-, LCMV- or murine sepsis-induced epigenetic-mediated immune suppression. A third clinically feasible strategy for reversing detrimental epigenetic scars includes bioengineering approaches to either directly reverse the detrimental epigenetic marks or to modify the epigenetic enzymes or transcription factors that induce detrimental epigenetic scars. Each of these approaches, alone or in combination, have ablated or reversed detrimental epigenetic marks in in-vitro or in animal models; translational studies are now required to evaluate clinical applicability.

show abstract

“…By analyzing biases in transposon density in genes previously identified as "non-essential", we found 40 genes where the encoded proteins contained putative essential and non-essential domains. Using a CRISPR Interference (CRISPRi) 15 platform we developed for Burkholderia 16 , we experimentally confirmed growth defects, representing the loss of an essential function, in 27 EDC gene knockdowns. The identified EDC genes include ten encoding known multidomain proteins and two entirely uncharacterized genes encoding different N-terminal DUFs, demonstrating the utility of the approach.…”

Section: Introductionmentioning

confidence: 81%

“…CRISPRi comprises a chromosomally integrated dCas9 under the control of a rhamnoseinducible promoter and plasmid-borne sgRNA driven by a constitutively active synthetic promoter, PJ23119 16 . Simultaneous expression of dCas9 and a target-specific sgRNA allows the dCas9 to bind the target DNA region and, thus, sterically interfere with transcription by RNA polymerase 15,16 . To inhibit the expression of the candidate genes, we designed two sgRNAs against each of the candidate genes targeting the start codon and adjacent region on the nontemplate strand (Supplementary Figure 3a and c).…”

Section: Crispri Knockdowns Of Edc Genes Show Growth Defectsmentioning

confidence: 99%

Identification of Essential Protein Domains From High-density Transposon Insertion Sequencing

Az¹,

Timmerman²,

Gallardo³

et al. 2021

Preprint

View full text Add to dashboard Cite

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term “essential” to whole genes rather than the protein domain sequences that confer the essential functions. However, genes can code for multiple protein domains that evolve their functions independently. Therefore, when essential genes code for more than one protein domain, only one of them could be essential. In this study, we defined this subset of genes as “essential domain-containing” (EDC) genes. Using a Tn-seq data set built-in Burkholderia cenocepacia K56-2, we developed an in silico pipeline to identify EDC genes and the essential protein domains they encode. We found forty candidate EDC genes and demonstrated growth defect phenotypes using CRISPR interference (CRISPRi). This analysis included two knockdowns of genes encoding the protein domains of unknown function DUF2213 and DUF4148. These essential domains are conserved in more than two hundred bacterial species, including human and plant pathogens. Together, our study suggests that essentiality should be assigned to individual protein domains rather than genes, contributing to a first functional characterization of protein domains of unknown function.

show abstract

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression

Cited by 113 publications

References 0 publications

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

Reversing Post-Infectious Epigenetic-Mediated Immune Suppression

Identification of Essential Protein Domains From High-density Transposon Insertion Sequencing

Contact Info

Product

Resources

About