Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants

Kiattisewee, Cholpisit; Karanjia, Ava V; Legut, Mateusz; Daniloski, Zharko; Koplik, Samantha E; Nelson, Joely; Kleinstiver, Benjamin P.; Sanjana, Neville E.; Carothers, James M.; Zalatan, Jesse G.

doi:10.1021/acssynbio.2c00405

Cited by 12 publications

(12 citation statements)

References 49 publications

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“…The heat shock–inducible system optimized in this study enabled the transient, temporal, and tunable regulation of target gene expression at different developmental stages following an induction by heat stress. To implement programmable gene regulation (Alon, 2007; Kiattisewee et al., 2022), extending our IGE system to CRISPR/dCas9(dead Cas9)‐based transcriptional and epigenetic regulation may be a good choice. Combining different inducible promoters, different heat‐shock conditions, and dCas9‐based transcriptional and epigenetic regulation may provide a facile method to regulate gene expression in a graded manner.…”

Section: Discussionmentioning

confidence: 99%

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

Liang,

Wei,

et al. 2023

The Plant Genome

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Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss‐of‐function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock–inducible genome‐editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat‐inducible promoter from heat‐shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat–inducible promoter heat shock–response element HSE‐COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat‐shock treatments at the seedling and bolting stages. A real‐time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE‐COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid‐insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE‐COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock–IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.

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Section: Discussionmentioning

confidence: 99%

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

Liang,

Wei,

et al. 2023

The Plant Genome

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“…Our results here suggest that dCas13 mediated activation (tlCRISPRa) could offer advantages over DNA-targeting dCas9 (txCRISPRa) by enabling control of individual genes in multi-gene operons. txCRISPRa is also subject to relatively stringent target requirements that have limited its generalizability to arbitrary gene targets (19, 58, 59). If tlCRISPRa with dCas13 is subject to less stringent or even simply different target requirements, it will expand the range of accessible gene targets in bacteria.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

Cardiff,

Faulkner,

Beall

et al. 2023

Preprint

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Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. InE. coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of both genes. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find that dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.Graphical Abstract

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“…macacae) or SmuCas9 (from Simonsiella muelleri) . Currently, the two most PAM-less variants, SpRY and SpdNG-LWQT, can recognize all PAMs, yet both exhibited compromised overall activities and lesser efficiency on NYN PAMs (where Y refers to T and C). , …”

Section: Introductionmentioning

confidence: 99%

“…25 Currently, the two most PAMless variants, SpRY 8 and SpdNG-LWQT, 11 can recognize all PAMs, yet both exhibited compromised overall activities and lesser efficiency on NYN PAMs (where Y refers to T and C). 26,27 In this study, we sought to generate robust PAM-less Cas9 variants with higher activities by exploring a Cas9 ortholog that demonstrated the potential to accommodate the most diverse PAM range among all the SpCas9-like orthologs in our previous study. 23 This Cas9 ortholog from S. equinus HC5 (SeHCas9) possesses an unprecedented RxQ PAM-binding motif within the PAM-interaction (PI) domain (Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Teng,

Wang,

Jiang

et al. 2023

ACS Synth. Biol.

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The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.

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Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants

Cited by 12 publications

References 49 publications

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

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