Protocol : Improved vectors and genome-wide libraries for CRISPR screening

Article summary

Genome-wide, targeted loss-of-function pooled screens using the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi). Previously, we used a genome-scale CRISPR knockout (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, thereby limiting the range of biological systems amenable to screening.

Reference: Sanjana, Neville E., Ophir Shalem, and Feng Zhang. "Improved vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784.

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Protocol : A CRISPR/Cas9 toolkit for multiplex genome editing in plants

Article summary

Background

To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. 

 Results

We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.  

Conclusions

We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Reference:Xing, Hui-Li, et al. "A CRISPR/Cas9 toolkit for multiplex genome editing in plants." BMC plant biology 14.1 (2014): 327.

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COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

Article summary

Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu). Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.

CRISPR design tools : CLICK HERE

Reference:Cradick, Thomas J., et al. "COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites." Molecular Therapy—Nucleic Acids 3.12 (2014): e214.

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CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

Summary:

CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast.

Reference:Naito, Yuki, et al. "CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites." Bioinformatics (2014): btu743.

CRISPR design tools : CLICK HERE

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Gene editing: how to stay on-target with CRISPR

Abstract

Efficiently cutting a target sequence to effect a desired change in the genome is one gene-editing task. Knowing where else in the genome a tool might have made its mark is quite another.

Table: CRISPR off target prediction tools

CasOT	PKU Zebrafish Functional Genomics group, Peking University
CHOPCHOP	Harvard University
CRISPR Design	Feng Zhang, Massachusetts Institute of Technology
CRISPR Design tool	The Broad Institute of Harvard and MIT
CRISPR/Cas9 gRNA finder	Jack Lin, University of Colorado
CRISPRfinder	Christine Pourcel, Université Paris-Sud 11
E-CRISP	DKFZ German Cancer Research Center
CRISPR gRNA Design tool	DNA 2.0
PROGNOS	Gang Bao, Emory University/Georgia Institute of Technology
ZiFiT	Keith Joung, Massachusetts General Hospital

Reference:Vivien Marx. Gene editing: how to stay on-target with CRISPR. Nature Methods 11, 1021–1026 (2014)

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Mouse Genome Editing Using the CRISPR/Cas System.

Abstract

The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here authors provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models

Reference:Harms, Donald W., et al. "Mouse Genome Editing Using the CRISPR/Cas System." Current Protocols in Human Genetics (2014): 15-7.

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MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

Motivation

 Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose. 

 Results

 Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. Authors show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles. 

 Availability and Implementation 

 MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfind​er. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.

Reference:

Abby SS, Néron B, Ménager H, Touchon M, Rocha EPC (2014) MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems. PLoS ONE 9(10): e110726. doi:10.1371/journal.pone.0110726

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Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together these data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.

Reference:Li, Kai, et al. "Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System." PloS one 9.8 (2014): e105779.

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Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation

Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. In this article, authors created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. They also discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.

Reference:Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation." Nature biotechnology (2014).

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Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. This article present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. Authors prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, They genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.

Reference:Kim, Jong Min, et al. "Genotyping with CRISPR-Cas-derived RNA-guided endonucleases." Nature communications 5 (2014).

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An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. Authors developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. This group also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%, therefore, they further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

Reference:Zhu, Xiaoxiao, et al. "An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System." Scientific reports 4 (2014).

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Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, authors provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. Authors have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, group show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. These findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.

Reference: Fineran, Peter C., et al. "Degenerate target sites mediate rapid primed CRISPR adaptation." Proceedings of the National Academy of Sciences 111.16 (2014): E1629-E1638.

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Protocol: Targeted Mutagenesis in Model and Crop Plants Using the CRISPR/Cas system

Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system.

The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review, researchers from The Sainsbury Laboratory summarize and discuss recent applications of the CRISPR/Cas technology in plants.

Belhaj, Khaoula, et al. "Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system." Plant methods 9.1 (2013): 39.

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Nature Protocol Publishes Genome Editing in Rice and Wheat using the CRISPR/Cas System

Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing.Here scientists from Institute of Genetics and Developmental Biology (IGDB) describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1–2 weeks in protoplasts, and mutated rice plants can be generated within 13–17 weeks.

Shan, Qiwei, et al. "Genome editing in rice and wheat using the CRISPR/Cas system." Nature protocols 9.10 (2014): 2395-2410.

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