Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity

Liang, Fu‐Sen; Ho, Wen Qi; Crabtree, Gerald R.

doi:10.1126/scisignal.2001449

Cited by 252 publications

(257 citation statements)

References 44 publications

Supporting

Mentioning

253

Contrasting

Unclassified

Order By: Relevance

“…S6). Fusing both lobes to domains that selectively dimerize upon chemical or optical induction, such as the abscisic acid-inducible PYL-ABI dimer (17) or the blue light-inducible CRY2-CIB1 dimer (18), would allow for enhanced spatiotemporal control of genome-engineering events (6). Dimerization domains may also increase the efficiency of complex formation by making lobe assembly independent of the sgRNA.…”

Section: Discussionmentioning

confidence: 99%

Rational design of a split-Cas9 enzyme complex

Wright

Sternberg²,

Taylor

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

273

207

View full text Add to dashboard Cite

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.CRISPR-Cas9 | genome engineering | split enzyme B acteria use RNA-guided adaptive immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) genomic loci to defend against invasive DNA (1, 2). In type II CRISPR-Cas systems, a single enzyme called Cas9 is responsible for targeting and cleavage of foreign DNA (3). The ability to program Cas9 for DNA cleavage at sites defined by engineered single-guide RNAs (sgRNAs) (4) has led to its adoption as a robust and versatile platform for genome engineering (for recent reviews, see refs. 5-7).Cas9 contains two nuclease active sites that function together to generate DNA double-strand breaks (DSBs) at sites complementary to the 20-nt guide RNA sequence and adjacent to a protospacer adjacent motif (PAM). Structural studies of the Streptococcus pyogenes Cas9 showed that the protein exhibits a bilobed architecture comprising the catalytic nuclease lobe and the α-helical lobe of the enzyme (8). Electron microscopy (EM) studies and comparisons with X-ray crystal structures with and without a bound guide RNA and target DNA revealed a largescale conformational rearrangement of the two lobes relative to each other upon nucleic acid binding (8, 9). Strikingly, RNA binding induces the nuclease lobe to rotate ∼100°relative to the α-helical lobe, generating a nucleic-acid binding cleft that can accommodate DNA, and interactions between the two lobes seem to be mediated primarily through contacts with the bound nucleic acid rather than direct protein-protein contacts (8, 9). These observations suggested that the two structural lobes of Cas9 might be separable into independent polypeptides that retain the ability to assemble into an active enzyme complex. Such a system would enable analysis of the functionally distinct properties of each Cas9 structural region and might offer a unique mechanism for control...

show abstract

Section: Discussionmentioning

confidence: 99%

Rational design of a split-Cas9 enzyme complex

Wright

Sternberg²,

Taylor

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

273

207

View full text Add to dashboard Cite

show abstract

“…mammalian-based orthogonal chemically induced dimerization (CID) systems (Liang et al, 2011;Miyamoto et al, 2012). Taken together, target-based screens offer the possibility to identify a small molecule ligand for a specific protein of interest and to further improve on small molecule binding.…”

Section: Target-based Screens and Synthetic Biologymentioning

confidence: 99%

Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology

Dejonghe

Russinova²

2017

Plant Physiol.

View full text Add to dashboard Cite

“…For example, two CID systems have been constructed on the basis of hormone signaling in plants; one involves the dimerization of the proteins gibberellin insensitive dwarf 1 (GID1) and gibberellin insensitive (GAI) upon the addition of the hormone gibberellin (Miyamoto et al, 2012); the other utilizes the plant hormone abscisic acid (ABA) to induce dimerization between the PYR1-like (PYL) and ABA insensitive 1 (ABI1) proteins (Liang et al, 2011). In another variation, a kinase-inducible biomolecular switch (KIBS) was used to induce dimerization.…”

Section: Genetically Encoded Tools That Can Be Used To Perturb Biochementioning

confidence: 99%

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

Sample

Mehta

Zhang

2014

Journal of Cell Science

View full text Add to dashboard Cite

In this Commentary, we discuss two sets of genetically encoded molecular tools that have significantly enhanced our ability to observe and manipulate complex biochemical processes in their native context and that have been essential in deepening our molecular understanding of how intracellular signaling networks function. In particular, genetically encoded biosensors are widely used to directly visualize signaling events in living cells, and we highlight several examples of basic biosensor designs that have enabled researchers to capture the spatial and temporal dynamics of numerous signaling molecules, including second messengers and signaling enzymes, with remarkable detail. Similarly, we discuss a number of genetically encoded biochemical perturbation techniques that are being used to manipulate the activity of various signaling molecules with far greater spatial and temporal selectivity than can be achieved using standard pharmacological or genetic techniques, focusing specifically on examples of chemically driven and light-inducible perturbation strategies. We then describe recent efforts to combine these diverse and powerful molecular tools into a unified platform that can be used to elucidate the molecular details of biological processes that may potentially extend well beyond the realm of signal transduction.

show abstract

Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity

Cited by 252 publications

References 44 publications

Rational design of a split-Cas9 enzyme complex

Rational design of a split-Cas9 enzyme complex

Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

Contact Info

Product

Resources

About