Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Farboud, Behnom; Meyer, Barbara J

doi:10.1534/genetics.115.175166

Cited by 226 publications

(237 citation statements)

References 48 publications

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“…To verify that rbm-5 mutations can suppress the ts-lethality of uaf-1(n4588) mutants, we used a modified CRISPR/Cas9 method [41] to generate new mutations in rbm-5 (Figure 3(a), sgRNA1, sgRNA2, sgRNA3 ). By this approach, we obtained five deletion mutations in rbm-5 (Figure 3(a) and Table S2).…”

Section: Resultsmentioning

confidence: 99%

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RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

Zhou

Gao

et al. 2018

RNA Biology

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A key step in pre-mRNA splicing is the recognition of 3ʹ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3ʹ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

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Section: Resultsmentioning

confidence: 99%

“…We followed the method by Farboud et al [41] with minor modifications. Plasmids for microinjection were purified using OMEGA’s Midi Plasmid Purification kit (Omega Bio-tek).…”

Section: Methodsmentioning

confidence: 99%

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

Zhou

Gao

et al. 2018

RNA Biology

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“…But short arms also mean that the cut site must be very close to the targeted insertion site (in our experiments separated by only 4-20 bp), which limits selection of the guide RNA binding site. The Meyer group recently demonstrated that guide RNAs that bind to sites with a diguanine motif immediately preceding the PAM sequence (called ggNGG guides) are significantly more efficient at generating double-stranded breaks in the worm genome in vivo (Farboud and Meyer 2015). Binding sites containing the ggNGG motif are significantly less common than binding sites containing only the NGG PAM sequence.…”

Section: Discussionmentioning

confidence: 99%

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

2016

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In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cellspecific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3-to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.KEYWORDS CRISPR/Cas9; high-throughput; gene tagging; plasmid toolkit T HE clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized genome editing in nearly all model systems, including Caenorhabditis elegans (Frøkjaer-Jensen 2013;Doudna and Charpentier 2014;Xu 2015). The Cas9 protein cuts genomic DNA at sites that match an 20-nucleotide guide RNA sequence (Gasiunas et al. 2012;Jinek et al. 2012). Error-prone repair of the break can create point mutations, small indels, or large deletions at the cut site (Cho et al. 2013;Cong et al. 2013;Friedland et al. 2013;Jinek et al. 2013;van Schendel et al. 2015). However, the true power of CRISPR/Cas9 for genome editing lies in the insertion of exogenous DNA sequences, such as genetically encoded protein tags (Katic and Grosshans 2013;Lo et al. 2013;Mali et al. 2013;Tzur et al. 2013). To insert an exogenous sequence, one must simply supply a repair template with homology arms that flank the cut site. The cell uses homologybased repair to heal the break and copy the exogenous DNA into the cut site.Although in theory CRISPR/Cas9 makes it easy to insert exogenous sequences into a genome, practical limitations have prevented high-throughput implementation. Two critical limiting factors are (1) the time and expense required to build both the repair template a...

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“…For instance, a highest cleavage efficiency was observed in C. elegans when the final 6 bp of the protospacers of Sp-derived sgRNAs contained 4 or more GCs [39]. Identically, a better frequency of cleavage and homology-driven gene knock-in was demonstrated in C. elegans with protospacers ending with a GG motif [40]. This could be due to the fact that a high proportion of GCs close to the PAM would make the Cas9 stay longer on the target and give it more chance of cutting.…”

Section: Selection Of Target Sitesmentioning

confidence: 97%

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Collonnier

Guyon‐Debast

Maclot

et al. 2017

Methods

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a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

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Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Cited by 226 publications

References 48 publications

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

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