A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9

Maji, Basudeb; Gangopadhyay, Soumyashree A.; Lee, Miseon; Shi, Mengchao; Wu, Peng; Heler, Robert; Mok, Beverly; Lim, Donghyun; Siriwardena, Sachini U.; Paul, Bishwajit; Dančík, Vlado; Vetere, Amedeo; Mesleh, Michael F.; Marraffini, Luciano A.; Liu, David R.; Clemons, Paul A.; Wagner, Bridget K.; Choudhary, Amit

doi:10.1016/j.cell.2019.04.009

Cited by 148 publications

(163 citation statements)

References 52 publications

(51 reference statements)

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“…Finally, to investigate the off-target profile of the Cas9-adaptor conjugates, we used an eGFP disruption assay with matched gRNA and mismatched gRNAs targeting the eGFP gene, in U2OS.eGFP.PEST cells. 30,31 The Cas9-adaptor conjugate retained the target specificity while maintaining the on-target activity ( Figure S4).…”

Section: Resultsmentioning

confidence: 96%

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

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Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC MAINCRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. 1 Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; 2,3 however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. For example, precise sequence alteration at the Cas9 cleavage site requires the efficient incorporation of exogenously supplied single-stranded oligonucleotide donor DNA (ssODN) 4 via the homology-directed repair (HDR) pathway. 5-7 However, most cells instead employ the nonhomologous end-joining (NHEJ) repair, which results in unpredictable insertions and deletions of bases at the cleavage site, some of which are large enough to have pathogenic consequences. 3,8,9 Displaying ssODNs on Cas9 can increase their local concentration around the DNA strand break site to allow enhanced incorporation of the desired sequence. In another application, appending PEG chains to Cas9 may reduce the immunogenicity of this bacterial protein. 10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display. Second, the platform should be robust and implementable by nonspecialists, given the diverse u...

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Section: Resultsmentioning

confidence: 96%

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

Self Cite

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“…Despite the elusive biological relevance of the anti-CRISPR activity of G8P, we nevertheless demonstrated that the genome-editing activity of SpyCas9 in human cells can be modulated by G8PPD. To the best of our knowledge, our discovery represents the first peptides known to exhibit Cas9-inhibiting activity and expands the anti-CRISPR agent toolbox which is currently composed of anti-CRISPR proteins [21], smallmolecules [20] and synthetic oligonucleotides [59]. Compared with Acr proteins that are typically 10 to 20 kD in size, G8PPD peptides are small in size and can be chemically proposed that the specificity of SpyCas9 in human cells may be increased by partial inhibition with weak CRISPR-Cas inhibitors [20].…”

Section: Discussionmentioning

confidence: 99%

“…To the best of our knowledge, our discovery represents the first peptides known to exhibit Cas9-inhibiting activity and expands the anti-CRISPR agent toolbox which is currently composed of anti-CRISPR proteins [21], smallmolecules [20] and synthetic oligonucleotides [59]. Compared with Acr proteins that are typically 10 to 20 kD in size, G8PPD peptides are small in size and can be chemically proposed that the specificity of SpyCas9 in human cells may be increased by partial inhibition with weak CRISPR-Cas inhibitors [20]. In the current study, we found that transfection of strong CRISPR-Cas inhibitor AcrII4A inhibited SpyCas9 activity to near complete in human cells when transfected prior to ( Fig.…”

Section: Discussionmentioning

confidence: 99%

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Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

Cui

Wang

et al. 2019

Preprint

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Background CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system.Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpyCas9) proteins in an allosteric manner. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpyCas9 while retaining its on-target activity. ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

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“…It might be possible to use HDAC3 inhibitors for turning down the activity of Cas9. The first small molecular Cas9 inhibitors have been uncovered recently which can be used for inactivating Cas9 activity34 . Although those inhibitors are with high potency, they might be only effective for SpCas9.…”

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confidence: 99%

Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

Liu

Chen

Rose

et al. 2019

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Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications.One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR.Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

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A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9

Cited by 148 publications

References 52 publications

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

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