A split CRISPR–Cpf1 platform for inducible genome editing and gene activation

Nihongaki, Yuta; Otabe, Takahiro; Ueda, Yoshibumi; Sato, Moritoshi

doi:10.1038/s41589-019-0338-y

Cited by 74 publications

(61 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each fragment is then fused to a partner domain such that the split protein is reconstituted, and its function is restored only when the partner domains interact. This modular strategy may be applied to diverse functional proteins to control bioluminescence 5, 6 , fluorescence 7 , proteolytic cleavage 8–10 and transcription 11, 12 . As a result, conditionally-reconstituted split proteins have been employed in a variety of applications including probing and discovering new protein-protein interactions 13–16 , studying post-translational modifications 17 , imposing small molecule-regulated control over enzymatic activity 18, 19 , and rewiring cellular signaling 9, 20 .…”

Section: Introductionmentioning

confidence: 99%

Computation-guided optimization of split protein systems

Dolberg¹,

Meger²,

Boucher³

et al. 2019

Preprint

View full text Add to dashboard Cite

25Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting 26 fragments is a powerful strategy for building tools to study and control biochemical systems. However, split 27 proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which 28 limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or 29 often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental 30 protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants-31 which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic 32 activity-to identify an optimal functional window. We validate our method by solving two distinct split 33 protein design challenges, generating both broad insights and new technology platforms. This method will 34 streamline the generation and use of split protein systems for diverse applications. 35 36 KEYWORDS: synthetic biology, split proteins, computational protein design, protein engineering 37 38

show abstract

Section: Introductionmentioning

confidence: 99%

Computation-guided optimization of split protein systems

Dolberg¹,

Meger²,

Boucher³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Cas12a has been demonstrated to process up to 25 crRNAs from a single transcript in the literature 47 , indicating that CRISPR/dCas12a systems may have the potential to extend multiplexing to target whole BGCs from a single crRNA array in future works. Additionally, next generation activators 48 could improve dCas12a-based CRISPRa activation strength.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Roux

Woodcraft

et al. 2020

Preprint

View full text Add to dashboard Cite

7Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low 8 expression of many biosynthetic gene clusters (BGCs) under standard culture conditions. In 9 this work, we develop a fungal CRISPR activation (CRISPRa) system for targeted upregulation 10 of biosynthetic genes, which could accelerate the emerging genomics-driven approach to 11 bioactive secondary metabolite discovery. We construct a fungal CRISPR/dLbCas12a-VPR 12 system and demonstrate activation of a fluorescent reporter in Aspergillus nidulans. Then, we 13 target the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both 14 chromosomal and episomal contexts, achieving increases in production of the compound 15 microperfuranone. Finally, multi-gene CRISPRa leads to the discovery of the mic cluster 16 product as dehydromicroperfuranone. Additionally, we demonstrate the utility of the variant 17 LbCas12a D156R -VPR for CRISPRa at lower culture temperatures. This is the first 18 demonstration of CRISPRa in filamentous fungi, providing a framework for CRISPR-mediated 19 transcriptional activation of fungal BGCs. 20 activation (CRISPRa)-mediated approach for BGC activation in filamentous fungi (Fig. 1a). In 49 CRISPRa systems, DNase-deactivated RNA-guided CRISPR/dCas ribonucleoprotein 50 complexes linked to activation effectors are targeted to gene regulatory regions to increase 51 gene expression [17][18][19] . Taking advantage of the streamlined CRISPR RNA (crRNA) cloning 52 and multiplexing capabilities, CRISPRa of BGCs has the potential to greatly accelerate fungal 53 genome mining. CRISPRa has already been used to tune the expression of biosynthetic 54 pathways 20,21 , including in ascomycetous yeasts 22,23 . However, to our knowledge, CRISPRa 55has not yet been demonstrated in filamentous fungi. 56In this work, we develop a suite of fungal CRISPRa vectors based on both dLbCas12a from 57Lachnospiraceae bacterium Cas12a (previously known as Cpf1) 19 , and dSpCas9 from 58Streptococcus pyogenes fused to the tripartite VPR activator 18 , and test them in A. nidulans. 59We further explore the application of our CRISPR/dLbCas12a-VPR system for fungal BGC 60 activation as a tool to fuel bioactive molecule discovery. 61 Results 62 Construction and testing of fungal CRISPRa systems 63To develop a CRISPRa system for filamentous fungi, we constructed and tested 64 CRISPR/dLbCas12a-VPR-and CRISPR/dSpCas9-VPR-based systems in the model 65 organism and chassis A. nidulans. To evaluate alternative strategies for expressing either 66 3 dCas effector, we created parent strains with a chromosomally integrated dCas-VPR 67 expression cassette and compared their performance with entirely AMA1-episomally encoded 68 systems. The AMA1 sequence acts as an extrachromosomal vector replicator and confers 69 increased transformation frequency in several filamentous fungi species 24 . AMA1-bearing 70 vectors are found at multiple copies per nucleus although their genetic stability has been 71 reported to be limited under non...

show abstract

“…For example, an optimizing CRISPR-Cas12a system realized seamless DNA editing in one pot (Wang et al, 2019). And a pair of split Cas12a and deficient/dead Cas12a (dCas12a) fragments showed potent efficiency in both rapamycin-inducible, photoactivatable genome editing and endogenous gene activation (Nihongaki et al, 2019). Recently Cas13 systems are further explored to more precisely cleave virus RNA (Freije et al, 2019) and track RNA dynamically (Yang et al, 2019).…”

Section: Synthetic Crispr-cas System Improves High-throughput Identifmentioning

confidence: 99%

Synthetic Biology Speeds Up Drug Target Discovery

Xie

Yang

et al. 2020

Front. Pharmacol.

View full text Add to dashboard Cite

As a rising emerging field, synthetic biology intends to realize precise regulations of cellular network by constructing artificial synthetic circuits, and it brings great opportunities to treat diseases and discover novel drug targets. Depending on the combination mode of different logic gates, various synthetic circuits are created to carry out multilevel regulations. In given synthetic circuits, drugs often act as inputs to drive circuits operation. It is becoming available to construct drug-responsive gene circuits for experimentally treating various disease models, including metabolic disease, immunity disease, cancer and bacterial infection. Synthetic biology works well in association with the CRISPR system for drug target functional screening. Remarkably, more and more well-designed circuits are developed to discover novel drug targets and precisely regulate drug therapy for diseases.

show abstract

A split CRISPR–Cpf1 platform for inducible genome editing and gene activation

Cited by 74 publications

References 37 publications

Computation-guided optimization of split protein systems

Computation-guided optimization of split protein systems

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Synthetic Biology Speeds Up Drug Target Discovery

Contact Info

Product

Resources

About