Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

He, Yuqing; Zhang, Tao; Yang, Ning; Xu, Meilian; Yan, Lang; Wang, Lihao; Wang, Rongchen; Zhao, Yunde

doi:10.1016/j.jgg.2017.08.003

Cited by 78 publications

(56 citation statements)

References 17 publications

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“…we first generated 44F03-3XgRNA#1: KD in R44F03-iKD1W (Awasaki et al, 2014) was replaced (HindIII/KpnI) with a 3XgRNA#1 cassette (synthetized by Genscript). This 3XgRNA#1 cassette contains three copies of a gRNA#1 flanked by the ribozymes HammerHead and HDV (He et al, 2017). We then digested this plasmid to generate two fragments: KpnI-3XgRNA#1-SgrAI and AgeI-3XgRNA#1-HindIII.…”

Section: Generation Of Transgenic Fly Lines A) Plasmid Generationmentioning

confidence: 99%

Unlimited genetic switches for cell-type specific manipulation

García-Marqués

Yang

Espinosa-Medina

et al. 2018

Preprint

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SummaryGaining independent genetic access to discrete cell types is critical to interrogate their biological functions, as well as to deliver precise gene therapy. Transcriptome analyses have allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types have remained inaccessible to genetic manipulation. Here, we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches that each respond to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell type-specific manipulations and combinatorial genetic targeting. CaSSA is thus a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

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Section: Generation Of Transgenic Fly Lines A) Plasmid Generationmentioning

confidence: 99%

Unlimited genetic switches for cell-type specific manipulation

García-Marqués

Yang

Espinosa-Medina

et al. 2018

Preprint

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“…Variant #1: a FseI-HH-gRN(#2)NA#1-HDV-HindIII fragment was de novo synthetized ( Genscript) . This contains a conditional gRNA#1 activatable by the gRNA#2 and flanked by the Hammerhead and HDV ribozymes (29). This fragment was then cloned into a FseI/HindIII site in DpnEE-pBPKD1Uw (Tzumin Lee lab).…”

Section: Methodsmentioning

confidence: 99%

CLADES: a programmable sequence of reporters for lineage analysis

García-Marqués

Yang

Espinosa-Medina

et al. 2019

Preprint

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We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a 7 technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic 8 switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES 9 to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth 10 order with reporter expression. This gives us temporal resolution of lineage development that can 11 be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. 12 When targeted to the germ line, the same cascade progresses across animal generations, marking 13 each generation with the corresponding combination of reporters. CLADES thus offers an 14 innovative strategy for making programmable cascades of genes that can be used for genetic 15 manipulation or to record serial biological events. 16 One Sentence Summary: A sequence of reporters for lineage analysis 17 Main Text: Cell lineage is an essential determinant in the acquisition of cell identity (1-2). 18 Establishing the association between cell lineage and fate is one of the fundamental challenges in 19 biology. Solving this puzzle will provide a unique framework to interrogate the molecular 20 mechanisms involved in cell type specification: it is not possible to fully understand how a 21 molecular factor affects cell fate decisions if we do not even know where/when these cell fate 22 decisions occur. 23While single-cell transcriptomics has made it possible to identify cell types with great detail, 24 tracing lineages in complex organisms remains challenging. Two main strategies have been 25 deployed: i) imaging-based methods that label cell lineages in intact tissues (3-4), and ii) DNA 26 sequencing-based methods which unravel cell lineages via phylogenetic analysis of DNA 27 mutations accumulated during development (5-7). Unless the specimen is accessible for real-time 28 visualization, imaging-based strategies are only able to label cell clones rather than tracing lineage 29 progression in a single individual. For organisms with stereotyped development, full lineages can 30 be assembled by resolving smaller segments in multiple individuals (8). Besides overlooking inter-31 individual differences, this approach is tedious and makes it impractical to analyze mutant lineages 32 in detail. On the other hand, methods based on DNA sequencing allow high-throughput analysis 33 of lineage progression, although the resolution is currently limited to major lineage branches (7). 34 Moreover, sequencing methods fail to recover spatial and morphological information, critical 35 features for identification of cell types and mutant phenotypes. 36 To circumvent these limitations, we developed CLADES, a technology based on CRISPR/Cas9 to 37 trace and manipulate lineages in Drosophila. Inspired by principles of synthetic biology, we 38 engineered a programmable system of genetic switches to control the activation and inactivation 39 of re...

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“…These gRNAs were selected based on their ON and OFF target scores (Benchling). Each of these gRNAs was preceded by an AttP site and a HammerHead ribozyme (33). Finally, a 3Xp3-RFP-polyA(a-tubulin) fragment was synthetized by PCR amplification, using pure genomic DNA from a fly line in which this cassette was used as a marker (Bloomington, #54590).…”

Section: Molecular Biologymentioning

confidence: 99%

CAMIO for deletion analysis of endogenous DNA sequences in multicellular organisms

Chen

Marqués

Sugino

et al. 2019

Preprint

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19The genome is the blueprint for an organism. Interrogating the genome, 20 especially locating critical cis-regulatory elements, requires deletion analysis. 21 This is conventionally performed using synthetic constructs, making it 22 cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed 23Mutagenesis of Individual Offspring (CAMIO) to achieve high-throughput analysis 24 of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate 25 independent deletions. Controlled by recombination, a single guide RNA is 26 stochastically chosen from a set targeting a specific DNA region. Combining two 27 sets increases variability, leading to either indels at 1-2 target sites or inter-target 28 deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-29 break repair, consequently creating offspring with diverse mutations. Thus, from 30 a single population cross, we can obtain a deletion matrix covering a large 31 expanse of DNA at both coarse and fine resolution. We demonstrate the ease 32 and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-33 transcriptional regulation. 34 35 36 non-physiological, prompting the search for tailor-made sequence-specific DNA 63 nucleases that permit targeted mutagenesis of native sequences. 64 65In the early 2000s, zinc-finger nucleases (ZFNs) 6 , the first of the tailored 66 nucleases, were heavily adopted 7,8 . Although it seemed a promising strategy for 67 genome editing, major setbacks, especially off-target toxicity limited its utility 9 . 68 Therefore, the enthusiasm for ZFNs declined after the arrival of transcription 69 activator-like effector nucleases (TALENs) 10-12 . The higher specificity of TALENs 70 led to fewer off-target disruptions, and hence less cytotoxicity 13 . However, 71 constructing a TALEN is technically challenging because the homologous 72 sequences encoding the TALEN repeats are prone to recombine with each 73 other 9 . 74 75The newest technology, CRISPR (clustered regularly interspaced short 76 palindromic repeats), was first exploited for targeted mutagenesis 14 and then 77 swiftly adopted to edit genomes of diverse organisms 15-20 . CRISPR's popularity 78 lies in its simplicity: a Cas9 nuclease plus an easily made guide RNA (gRNA) 79 induces a double-strand break (DSB) targeted by DNA-RNA base pairing 14,16 . 80Cells repair the DSBs either through nonhomologous end joining (NHEJ) 21 , 81 leading to insertion or deletion (indel), or through homology-directed repair 82 (HDR) 22 which replaces discontinuous sequences with an available template. 83The fact that CRISPR can produce targeted DNA modifications with near 84 unlimited specificity has ensured its rapid expansion throughout the biological 85 7 132 Figure 1. bamP-Cas9 induces efficient CRISPR targeted mutagenesis in male germ 133 cells. (A) Ebony transcript shown with UTRs in turquoise. U6 drives guide RNA (gRNA-134 e), which targets 5' end of ebony coding sequence. (B) Percent of progeny with ebony 135 loss-of-function (LOF) mutati...

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Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

Cited by 78 publications

References 17 publications

Unlimited genetic switches for cell-type specific manipulation

Unlimited genetic switches for cell-type specific manipulation

CLADES: a programmable sequence of reporters for lineage analysis

CAMIO for deletion analysis of endogenous DNA sequences in multicellular organisms

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