Guide RNA engineering for versatile Cas9 functionality

Nowak, Chance M.; Lawson, Seth; Zerez, Megan; Bleris, Leonidas

doi:10.1093/nar/gkw908

Cited by 62 publications

(59 citation statements)

References 94 publications

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“…For scRNA and SAM, EDLL was attached to dCas9 and VP64 was fused to the MS2 viral coat protein, which binds an RNA aptamer. RNA scaffolds in scRNA and SAM designs contained a second optimized aptamer next to the wild type one, as this double-aptamer design was earlier found to improve binding activity 18 . During the cloning of the gRNA2.0 scaffold employed in the scRNA design, a spontaneous mutation occurred consisting in the insertion of an adenine in the loop of the first aptamer ( Supplementary Figure 2).…”

Section: Resultsmentioning

confidence: 99%

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator

Selma

Bernabé‐Orts

Vázquez‐Vilar

et al. 2018

Preprint

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Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous activation domains (ADs) capable of recruiting the cell´s transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated Cas9 protein (dCas9), creating so-called Programmable Transcriptional Activators (PTAs). In search for optimized dCas9-PTAs we performed a combinatorial analysis with seven different ADs arranged in four different protein/RNA architectures. This analysis resulted in the selection of a new dCas9-PTA with improved features as compared with previously reported activators. The new synthetic riboprotein, named dCasEV2.1, combines EDLL and VPR ADs using a multiplexable mutated version (v2.1) of the previously described aptamer-containing guide RNA2.0. We show here that dCasEV2.

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Section: Resultsmentioning

confidence: 99%

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator

Selma

Bernabé‐Orts

Vázquez‐Vilar

et al. 2018

Preprint

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“…Less than ten colonies were obtained on the plate, possibly due to the high selection pressure of 5'-FOA and the slow cleavage activity of Cpf1: majority of cells were killed by 5'-FOA before they could be edited by Cpf1. It has been recently discovered that the addition of poly-thymidine to the 3'-end of crRNA could improve the AsCpf1 genome editing efficiency (Xie, Minkenberg et al 2015, Nowak, Lawson et al 2016, Bin Moon, Lee et al 2018. We thus modified the crRNA by adding eight thymidine (T8), which will form a poly-U overhang following the 3'-end of the crRNA.…”

Section: Crispr-cpf1 Mediated In-del Mutations Of Counter-selectable mentioning

confidence: 99%

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing inYarrowia lipolytica

Yang

Edwards

2019

Preprint

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CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved highediting efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Y. lipolytica: arginine permease (93% for CAN1) and orotidine 5'-phosphate decarboxylase (~96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur housekeeping genetic markers (40%-75% for MET2, MET6 and MET25), which forms distinct color changes in yeast colonies due to the precipitation of PbS (lead sulfide). Different from Cas9, we demonstrated that the crRNA transcribed from a type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3' polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%-83% for duplex genomic targets and 41.7% for triplex genomic targets. Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast cell factories for various applications.

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“…Spatiotemporal control has been achieved by regulation of Cas9 via photoactivation 17 or via tissue-specific promoters 18,19 or microRNAs, 20 which comes with the unwelcome restriction that all gRNAs are subject to the same regulatory scope. Systematic mapping of the structure and sequence properties of functional gRNAs has revealed that Cas9 activity is tolerant to significant modifications the standard gRNA structure, 21,22 facilitiating introduction of auxiliary domains that enable conditional control of gRNA activity via structural changes induced by small-molecules, 23,24 protein-bound RNAs, 25 nucleases, 26 or nuclease-recruiting DNAs. 26 Alternatively, the activity of standard gRNAs has been modulated by antisense RNAs 27 or by photolysis of antisense DNAs incorporating photocleavable groups.…”

Section: Introductionmentioning

confidence: 99%

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Hanewich-Hollatz

Chen

Huang

et al. 2019

Preprint

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Dynamic RNA Nanotechnology in Living Cells Programmable RNA trigger X dCas9 Constitutively inactive (OFF ON logic) cgRNA Y Programmable DNA target Constitutively active (ON OFF logic) X dCas9 Y cgRNAA guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON→OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF→ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe programmable conditional gene silencing with a median dynamic range of ≈6-fold for an ON→OFF "terminator switch" mechanism, ≈15-fold for an ON→OFF "splinted switch" mechanism, and ≈3.6-fold for an OFF→ON "toehold switch" mechanism; the median crosstalk within each cgRNA library is <2%, <2%, and ≈20% for the three mechanisms. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

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Guide RNA engineering for versatile Cas9 functionality

Cited by 62 publications

References 94 publications

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing inYarrowia lipolytica

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

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