Protocols for the CRISPR/Cas Technology

flySAM Transgenic CRISPRa System Manual

YJ Yu Jia

DS Da Shen

XW Xia Wang

JS Jin Sun

PP Ping Peng

RX Rong-Gang Xu [...]

Jian-Quan Ni ^*

+ 1 Author

0 Q&A

3 Favorites

8961 Views

Jan 20, 2019

Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.

CRISPR-Cas9 Mediated Genome Editing in <em>Drosophila</em>

CRISPR-Cas9 Mediated Genome Editing in Drosophila

PP Ping Peng

XW Xia Wang

DS Da Shen

JS Jin Sun

YJ Yu Jia

RX Rong-Gang Xu [...]

Jian-Quan Ni ^*

+ 1 Author

0 Q&A

4 Favorites

10316 Views

Jan 20, 2019

In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.

<em>In vitro</em> Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells

In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells

NS Nataša Savić

FR Femke CAS Ringnalda

CB Christian Berk

KB Katja Bargsten

JH Jonathan Hall

MJ Martin Jinek

GS Gerald Schwank^*

0 Q&A

2 Favorites

10890 Views

Jan 5, 2019

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.

CRISPR/Cas9-mediated ssDNA Recombineering in <em>Corynebacterium glutamicum</em>

CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum

JL Jiao Liu

Yu Wang

Ping Zheng^*

Jibin Sun^*

0 Q&A

1 Favorites

7503 Views

Oct 5, 2018

Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are now revolutionizing genome editing. The CRISPR/Cas9 system from Streptococcus pyogenes that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. In this protocol, we described the general procedure for CRISPR/Cas9-mediated ssDNA recombineering in C. glutamicum. Small modifications can be introduced into the C. glutamicum chromosome with a high editing efficiency up to 90%.

CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus

MB Manuel V. Borca

KB Keith A. Berggren

E Elizabeth Ramirez-Medina

EV Elizabeth A. Vuono

Douglas P. Gladue^*

0 Q&A

1 Favorites

9164 Views

Aug 20, 2018

Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.

High Resolution Melting Temperature Analysis to Identify CRISPR/Cas9 Mutants from <em>Arabidopsis</em>

High Resolution Melting Temperature Analysis to Identify CRISPR/Cas9 Mutants from Arabidopsis

CD Cynthia Denbow

SE Sonia Carole Ehivet

SO Sakiko Okumoto^*

0 Q&A

2 Favorites

8398 Views

Jul 20, 2018

CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results in single-bp insertion or deletion. While single-bp insertions or deletions (indels) effectively destroy the function of protein-coding genes through frameshift, detection is difficult due to the small size shift. High-resolution melting temperature analysis allows quick detection, and it does not require any additional pipetting steps after the PCR amplification of the region of interest. In this protocol, we will describe the steps required for the analysis of potential homozygous mutants.

Single and Multiplexed Gene Editing in <em>Ustilago maydis</em> Using CRISPR-Cas9

Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9

Mariana Schuster^*

CT Christine Trippel

PH Petra Happel

DL Daniel Lanver

SR Stefanie Reißmann

RK Regine Kahmann^*

0 Q&A

1 Favorites

9006 Views

Jul 20, 2018

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.

Method for CRISPR/Cas9 Mutagenesis in <em>Candida albicans</em>

Method for CRISPR/Cas9 Mutagenesis in Candida albicans

ND Neta Dean^*

HN Henry Ng

0 Q&A

2 Favorites

12015 Views

Apr 20, 2018

Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days.

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Andrew M. Tidball^*

PS Preethi Swaminathan

LD Louis T. Dang

JP Jack M. Parent

1 Q&A

5 Favorites

11661 Views

Apr 5, 2018

For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.

Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9

AA Azat Akhmetov

JL Jon M Laurent

Jimmy Gollihar

EG Elizabeth C Gardner

RG Riddhiman K Garge

AE Andrew D Ellington [...]

EM Edward M Marcotte^*

+ 1 Author

1 Q&A

11 Favorites

19248 Views

Mar 20, 2018

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in <em>Toxoplasma gondii</em>

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii

SL Shaojun Long

KB Kevin M. Brown

LS L. David Sibley^*

0 Q&A

0 Favorites

11695 Views

Mar 20, 2018

Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites.

Synthetic Genetic Interaction (CRISPR-SGI) Profiling in <em>Caenorhabditis elegans</em>

Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans

JC John A. Calarco

AN Adam D. Norris^*

0 Q&A

0 Favorites

8206 Views

Mar 5, 2018

Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in C. elegans and replacing them with a heterologous fluorescent reporter (Norris et al., 2015). Recently we took advantage of that system to perform a large-scale, reverse genetic screen using null alleles in animals for the first time, focusing on RNA binding protein genes (Norris et al., 2017). This type of approach should be similarly applicable to many other gene classes in C. elegans. Here we detail the protocols involved in generating a library of double mutants and performing medium-throughput competitive fitness assays to test for genetic interactions resulting in fitness changes.

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in <em>Toxoplasma gondii</em>

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

KB Kevin M. Brown

SL Shaojun Long

LS L. David Sibley^*

0 Q&A

1 Favorites

17220 Views

Feb 20, 2018

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in <em>Escherichia coli</em>

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

EK Enrico König

FZ Francesca Zerbini

IZ Ilaria Zanella

DF Davide Fraccascia

GG Guido Grandi^*

1 Q&A

13 Favorites

27504 Views

Jan 20, 2018

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid that allows starting a new mutation after only two working days. Our protocol allows multiple, stepwise gene knockout strains with high mutagenesis efficiencies applicable for high-throughput approaches.

Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9

Marie-Laurence Lemay

Ariane C. Renaud

Geneviève M. Rousseau

Sylvain Moineau^*

1 Q&A

7 Favorites

15900 Views

Jan 5, 2018

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.

Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins

Yu Murakami

SA Satoshi Ansai

AY Akari Yonemura

MK Masato Kinoshita^*

0 Q&A

3 Favorites

7300 Views

Dec 20, 2017

This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.

Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions

AH Amanda Hopes^*

VN Vladimir Nekrasov

NB Nigel Belshaw

IG Irina Grouneva

SK Sophien Kamoun

TM Thomas Mock^*

1 Q&A

3 Favorites

13644 Views

Dec 5, 2017

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

Rubul Mout^*

VR Vincent M. Rotello^*

1 Q&A

3 Favorites

12567 Views

Oct 20, 2017

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users’ need.Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al., 2017). We report here only the generation of the delivery vehicle i.e., the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for <em>Escherichia coli</em>

Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli

XL Xin-tian Li

CS Cindy Sou

SJ Suckjoon Jun^*

0 Q&A

0 Favorites

10288 Views

Oct 5, 2017

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.

Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants

XT Xu Tang

ZZ Zhaohui Zhong

XZ Xuelian Zheng

YZ Yong Zhang^*

0 Q&A

5 Favorites

12985 Views

Sep 5, 2017

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing (Gheysen et al., 1990). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.

Using CRISPR-ERA Webserver for sgRNA Design

HL Honglei Liu

XW Xiaowo Wang^*

Lei S. Qi^*

0 Q&A

9 Favorites

12311 Views

Sep 5, 2017

The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/) is one of the state-of-the-art designer webserver tools, which has been developed both for gene editing and gene regulation sgRNA design. This protocol discusses how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.

Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting

Fuqiang Chen^*

XD Xiao Ding

YF Yongmei Feng

TS Timothy Seebeck

YJ Yanfang Jiang

GD Gregory D Davis

0 Q&A

2 Favorites

9988 Views

Aug 5, 2017

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro (Knight et al., 2015; Hinz et al., 2015; Horlbeck et al., 2016; Isaac et al., 2016). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 (Chen et al., 2017). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.

CRISPR/Cas9 Gene Editing in the Marine Diatom <em>Phaeodactylum tricornutum</em>

CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum

Marianne Nymark^*

AS Amit Kumar Sharma

MH Marthe C. G. Hafskjold

TS Torfinn Sparstad

AB Atle M. Bones

PW Per Winge

0 Q&A

5 Favorites

13895 Views

Aug 5, 2017

The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.

Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System

MM Miriam Manor

Udi Qimron^*

0 Q&A

2 Favorites

11085 Views

Aug 5, 2017

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well.

Using CRISPR/Cas9 for Large Fragment Deletions in <em>Saccharomyces cerevisiae</em>

Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

HH Huanhuan Hao

JH Jing Huang

TL Tongtong Liu

HT Hui Tang

LZ Liping Zhang^*

0 Q&A

6 Favorites

13499 Views

Jul 20, 2017

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.

Generation of Targeted Knockout Mutants in <em>Arabidopsis thaliana</em> Using CRISPR/Cas9

Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

FH Florian Hahn

ME Marion Eisenhut

OM Otho Mantegazza

Andreas P. M. Weber^*

1 Q&A

16 Favorites

24622 Views

Jul 5, 2017

The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

YN Yiyuan Niu

YD Yi Ding

XW Xiaolong Wang

YC Yulin Chen^*

0 Q&A

1 Favorites

11036 Views

Jul 5, 2017

Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

Dense sgRNA Library Construction Using a Molecular Chipper Approach

JC Jijun Cheng^*

WP Wen Pan

JL Jun Lu^*

0 Q&A

0 Favorites

10157 Views

Jun 20, 2017

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.

Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses

TN Toshihisa Nomura^*

HS Hitoshi Sakakibara

0 Q&A

3 Favorites

12555 Views

Jun 20, 2017

RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID

TA Takayuki Arazoe

KN Keiji Nishida^*

AK Akihiko Kondo^*

0 Q&A

3 Favorites

19066 Views

Jun 5, 2017

Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an example, while this method principally should be applicable to any gene of interest in a wide range of cell types.Figure 1. Schematic illustration for Target-AID and its targetable site. In a guide-RNA (gRNA)-dependent manner, PmCDA1 fused to nCas9 (D10A) via a linker performs programmable cytidine mutagenesis around -21 to -16 positions relative to PAM sequence on the non-complementary strand in mammalian cells. The targetable site was determined based on the efficient base substitution (> 20%) observed in the previous work.

Expression and Purification of the Cas10-Csm Complex from Staphylococci

Lucy Chou-Zheng

Asma Hatoum-Aslan^*

0 Q&A

0 Favorites

11256 Views

Jun 5, 2017

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis and Staphylococcus aureus are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao et al., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al., 2014; Hatoum-Aslan et al., 2014; Samai et al., 2015) in these organisms. Thus, gaining a mechanistic understanding of these systems in the native staphylococcal background can lead to important insights into the factors that impact the evolution and survival of these pathogens. Type III-A CRISPR-Cas systems encode a five-subunit effector complex called Cas10-Csm (Hatoum-Aslan et al., 2013). Here, we describe a protocol for the expression and purification of Cas10-Csm from its native S. epidermidis background or a heterologous S. aureus background. The method consists of a two-step purification protocol involving Ni2+-affinity chromatography and a DNA affinity biotin pull-down, which together yield a pure preparation of the Cas10-Csm complex. This approach has been used previously to analyze the effects of mutations on Cas10-Csm complex integrity (Hatoum-Aslan et al., 2014), crRNA formation (Hatoum-Aslan et al., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker et al., 2016). Importantly, this approach can be easily adapted for use in other Staphylococcus species to probe and understand their native Type III-A CRISPR-Cas systems.

DNA-free Genome Editing of <em>Chlamydomonas reinhardtii</em> Using CRISPR and Subsequent Mutant Analysis

DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis

JY Jihyeon Yu

KB Kwangryul Baek

EJ EonSeon Jin^*

Sangsu Bae^*

0 Q&A

2 Favorites

15023 Views

Jun 5, 2017

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.

A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9

SH Satoshi Hara

MT Miho Terao

ST Shuji Takada^*

0 Q&A

1 Favorites

10758 Views

Jun 5, 2017

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of these RNAs less difficult when compared to sgRNA. Another downside of the CRISPR/Cas9 systems is that off-target effects have been reported. However, modified forms of Cas9 have been developed to minimize off-target effects. For example, nickase-type Cas9 (nCas9) and FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9) induce DSBs only when two guide RNAs bind opposite strands within a defined distance. In this protocol, we describe our experimental system for the production of mutant mice using a CRISPR/Cas9 system that combines crRNA, tracrRNA, and modified forms of Cas9. This method not only facilitates the preparation of reagents for the genome editing system but it can also reduce the risk of off-target effects.

Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors

Chin-kai Chuang^*

Ching-Fu Tu

Chien-Hong Chen

0 Q&A

2 Favorites

12074 Views

Jun 5, 2017

A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to generate gene-knockout pig.

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

BN Brooke A Napier

DM Denise M Monack^*

0 Q&A

7 Favorites

13123 Views

May 20, 2017

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in <em>Saccharomyces cerevisiae</em>

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Yu Sasano

SH Satoshi Harashima^*

0 Q&A

1 Favorites

10317 Views

May 20, 2017

Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four independent sites in the genome can be split by one transformation. Total time and labor for obtaining a multiple split yeast strain is drastically reduced when compared with conventional PCS technology.

Endogenous C-terminal Tagging by CRISPR/Cas9 in <em>Trypanosoma cruzi</em>

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Noelia Lander^*

Miguel A. Chiurillo

AV Aníbal E. Vercesi

Roberto Docampo

0 Q&A

2 Favorites

14901 Views

May 20, 2017

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism

Hiroshi Arakawa^*

0 Q&A

2 Favorites

10076 Views

May 20, 2017

While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA.Figure 1. A method to convert mRNA into a gRNA library construction (Sanjana et al., 2014). The scheme of the method is summarized. Each step of D-O is described in detail in the Procedure. Bg, BglII; Xb, XbaI; Bs, BsmBI; Aa, AatII. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2.

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

FW Forrest C. Walker

Asma Hatoum-Aslan^*

0 Q&A

3 Favorites

12305 Views

May 5, 2017

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

CRISPR/Cas9 Editing of the <em>Bacillus subtilis</em> Genome

CRISPR/Cas9 Editing of the Bacillus subtilis Genome

PB Peter E. Burby

LS Lyle A. Simmons^*

4 Q&A

10 Favorites

20650 Views

Apr 20, 2017

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed.

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

LB Lena Behrmann

Scott McComb

JA Júlia Aguadé-Gorgorió

YH Yun Huang

MH Mario Hermann

PP Pawel Pelczar [...]

Beat C. Bornhauser^*

+ 2 Authors

0 Q&A

6 Favorites

20042 Views

Apr 5, 2017

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery

GV Giel Vandemoortele

DD Delphine De Sutter

SE Sven Eyckerman^*

0 Q&A

4 Favorites

22201 Views

Apr 5, 2017

The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.

Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration

Kangquan Yin

TH Ting Han

Yule Liu^*

0 Q&A

5 Favorites

15081 Views

Apr 5, 2017

CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end joining (NHEJ) pathway (Wang et al., 2016). Since NHEJ is error prone, mutations are thus generated. In plants, delivery of genome editing reagents is still challenging. In this protocol, we detail the procedure of a virus-based gRNA delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE). This method offers a rapid and efficient way to deliver gRNA into plant cells, especially for those that are recalcitrant to transformation with Agrobacterium.

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

JG Jonathan M. Geisinger^*

Michele P. Calos

0 Q&A

2 Favorites

10262 Views

Mar 5, 2017

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in <em>Arabidopsis</em> by CRISPR-Cas9

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9

JS Julia Schumacher

KK Kerstin Kaufmann

WY Wenhao Yan^*

0 Q&A

7 Favorites

12162 Views

Mar 5, 2017

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana UBIQUITIN10 (UBQ10)-promoter and guide RNA (gRNA) expression cassettes driven by the A. thaliana U6-promoter for efficient multiplex editing in Arabidopsis (Yan et al., 2016). Here, we describe a step-by-step protocol to cost-efficiently generate the binary vector containing multiple gRNAs and the Cas9 nuclease based on classic cloning procedure.

Production of Guide RNAs <em>in vitro</em> and <em>in vivo</em> for CRISPR Using Ribozymes and RNA Polymerase II Promoters

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters

TZ Tao Zhang

YG Yangbin Gao

RW Rongchen Wang

YZ Yunde Zhao^*

0 Q&A

7 Favorites

17749 Views

Feb 20, 2017

CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.

Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in <em>Drosophila</em> Cells

Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells

BH Benjamin E. Housden^*

HN Hilary E. Nicholson

NP Norbert Perrimon^*

0 Q&A

1 Favorites

11118 Views

Feb 5, 2017

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

JV Johan Vad-Nielsen^*

Lin Lin ^*

KJ Kristopher Torp Jensen

AN Anders Lade Nielsen

Yonglun Luo

1 Q&A

12 Favorites

26467 Views

Dec 5, 2016

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter.

<em>In utero</em> Electroporation of Mouse Cerebellar Purkinje Cells

In utero Electroporation of Mouse Cerebellar Purkinje Cells

Yukari H. Takeo^*

0 Q&A

2 Favorites

15774 Views

Jun 5, 2016

In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System

KX Kabin Xie

Bastian Minkenberg

Yinong Yang^*

0 Q&A

11 Favorites

35415 Views

Sep 5, 2014

RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this protocol, we describe the general procedures for plant RGE using CRISPR-Cas9 system and Agrobacterium-mediated transformation. The protocol includes gRNA design, Cas9-gRNA plasmid construction and mutation detection (genotyping) for rice RGE and could be adapted for other plant species.

Identification of Proteins Interacting with Genomic Regions of Interest <em>in vivo</em> Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)

Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)

Toshitsugu Fujita

Hodaka Fujii^*

0 Q&A

1 Favorites

14819 Views

May 20, 2014

Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) for isolation of target genomic regions (Hoshino and Fujii, 2009; Fujita and Fujii, 2011; Fujita and Fujii, 2012; Fujita and Fujii, 2013a; Fujita and Fujii, 2013b; Fujita et al., 2013). Identification and characterization of molecules interacting with the isolated genomic regions facilitates understanding of molecular mechanisms of functions of the target genome regions. Here, we describe enChIP, in which engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and a catalytically inactive Cas9 (dCas9) plus small guide RNA (gRNA), are utilized for affinity purification of target genomic regions. The scheme of enChIP is as follows:1. A zinc-finger protein, TAL or dCas9 plus gRNA is generated to recognize DNA sequence in a genomic region of interest. 2. The engineered DNA-binding molecule is fused with a tag(s) and the nuclear localization signal (NLS), and expressed in the cell to be analyzed.3. The resultant cell is crosslinked, if necessary, and lysed, and DNA is fragmented.4. The complexes including the engineered DNA-binding molecule are subjected to affinity purification such as mmunoprecipitation. The isolated complexes retain molecules interacting with the genomic region of interest.5. Reverse crosslinking and subsequent purification of DNA, RNA, or proteins allow identification and characterization of these molecules.In this protocol, we describe enChIP with a TAL protein to isolate a genomic region of interest and analyze the interacting proteins by mass spectrometry (Fujita et al., 2013).