TECH NOTEImproved CRISPR/Cas9 Genome Editing inHard to Transfect Mammalian Cells UsingAAVAAV mediated delivery of Cas9 and sgRNA expression cassettes results in more indels, especiallyin hard to transfect cell lines:A higher rate of mutation is obtained with AAV mediated delivery of Cas9 and sgRNA compared totransfection based delivery An innovative method to deliver the large Cas9 gene with a small virus:To overcome the size limitation of AAV, the Cas9 gene into is divided between two vectors Viruses that encode truncated portions of Cas9 with a 1.6 kb region of homology are prepared in HEK293T packaging cells; recombination in target cells results in a full length Cas9 gene expressioncassette OverviewThe use of CRISPR/Cas9 technology can be limited by delivery options for Cas9 and the single guide RNA(sgRNA). Transfection of cells with plasmids encoding Cas9 and sgRNA is the most commonly used method.However, many human cell types are considered hard to transfect, making plasmid based delivery difficult. Onealternate strategy for delivering CRISPR/Cas9 components to these cell types is viral transduction.Advantages of AAV for CRISPR/Cas9 deliveryRecombinant adeno associated virus (AAV) has several advantages over other types of viruses for genedelivery. Importantly, AAV does not integrate into the host genome, precluding genomic integration andsustained expression of Cas9, thereby reducing the likelihood of Cas9 off target effects. AAV also exhibitslower immunogenicity and has a small genome relative to other non integrating viruses (e.g., adenovirus),making it easier to manipulate.An AAV based system for Cas9 and sgRNA deliveryThe small size of the AAV genome can be a limitation when packaging a large gene such asthe S.pyogenes Cas9 (SpCas9). The 4.1 kb SpCas9 gene together with its optimal promoter andpolyadenylation signal exceeds the capacity that can be efficiently packaged into AAV viral particles (Byrne etal., 2014). To overcome this limitation, we developed a system that takes advantage of DNA recombination thatis inherent to AAV genome processing (Hirsch et al., 2010).The vectors included with the AAVpro CRISPR/Cas9 Helper Free System (AAV2) split the Cas9 gene into twoportions (Cas9 Up and Cas9 Down) that have a 1.6 kb shared region of homology (Figure 1). This homologousregion mediates recombination with high efficiency inside the target cell, thereby producing a full length Cas9expression cassette.
Figure 1. AAV vector recombination to produce full length Cas9. The large size of the Cas9 gene (4.1 kb) precludesits packaging into a single AAV particle. The pAAV Guide it Up and pAAV Guide it Down plasmids contain truncatedupstream and downstream portions of Cas9 respectively, with a shared 1.6 kb region of homology. Separate viruses aregenerated with each plasmid. Transduction of target cells with both viruses results in recombination to generate a fulllength Cas9 expression cassette.Upstream and downstream portions of Cas9, with a shared 1.6 kb region of homology are split between twoseparate plasmids, pAAV Guide it Up and pAAV Guide it Down, respectively (Figure 2). A user defined guidesequence is cloned into the pAAV Guide it Down plasmid, which is provided prelinearized to allow for seamless,single step cloning (Figure 2B).
Figure 2. Vector maps of pAAV Guide it Up and pAAV Guide it Down. (A) The pAAV Guide it Up plasmid contains atruncated upstream portion of Cas9 (3,106 bp) that encodes the N terminal 1,035 amino acids of Cas9 under the controlof the CMV promoter. (B) The pAAV Guide it Down plasmid contains the downstream portion of Cas9 (2,616 bp) thatencodes the C terminal 872 amino acids, and is provided pre linearized. A gene specific guide sequence can becloned into the pAAV Guide it Down plasmid downstream of the human U6 promoter. Oligos encoding the sgRNA areannealed to form a duplex. The duplexed DNA is then cloned into the pre linearized pAAV Guide it Down plasmid.The AAVpro CRISPR/Cas9 Helper Free System workflowThe AAVpro CRISPR/Cas9 Helper Free System (AAV2) is a complete system for the delivery of Cas9 and agene specific sgRNA to mammalian cells using AAV2 (AAV serotype 2).First, oligos encoding a gene specific guide sequence are annealed to form duplexes. The duplexed DNA isligated to the pre linearized pAAV Guide it Down plasmid (Figure 2B). All the components needed for theannealing and ligation of the oligos are provided.AAV2 Up and AAV2 Down viruses are packaged independently in a HEK 293T packaging cell line. Packagingcells are transfected with the respective pAAV Guide it Up and pAAV Guide it Down plasmids, and the pHelperand pRC2 mi342 plasmids that encode viral packaging components and miRNA 342, a human microRNA thatincreases AAV2 titer. AAV2 viral particles are then extracted using the included AAVpro Extraction Solution.
Target cells are then transduced with equal number of AAV2 Up and AAV2 Down viruses to ensure efficientrecombination. Recombination between Cas9 Up and Cas9 Down within target cells results in a full length Cas9 gene expression cassette. Following transcription and translation, Cas9 is guided to the targetgenomic locus by the sgRNA, where it creates a double stranded break.Figure 3. Workflow of the AAVpro CRISPR/Cas9 Helper Free System (AAV2). The pAAV Guide it Up and pAAV Guide it Down vectors encode truncated upstream and downstream portions of the Cas9 gene respectively, with a 1.6 kb overlapping region of homology. Duplexed guide sequence oligos targeting a specific gene are ligated to the pre linearized pAAV Guide it Down plasmid. Separate AAV2 Up and AAV2 Down viruses are generated by transfectingHEK 293T cells with either pAAV Guide it Up or pAAV Guide it Down along with pRC2 mi342 and pHelper plasmids.The resulting viral particles are isolated using AAV Extraction Solution. Target cells are co transduced with equalnumber of both AAV2 Up and AAV2 Down viruses. The 1.6 kb homologous region results in recombination during AAV
genome processing giving rise to full length Cas9 that is transcribed and translated. The Cas9/sgRNA complex thenmediates cleavage of the target gene.Produce consistent and equivalent high titer AAV2 Up and AAV2 Down virusTo ensure efficient genome editing, obtaining high and equivalent titers of both AAV2 Up and AAV2 Down virusis critical. AAV2 Up and AAV2 Down viral particles encoding truncated portions of Cas9 and a sgRNAtargeting CCR5 were generated in three independent experiments using the AAVpro CRISPR/Cas9 Helper FreeSystem (AAV2) (Figure 4). Identically high titers were obtained for both viruses. The AAVpro ExtractionSolution, which is provided with the kit, results in viral yields that are 3 times higher than those obtained withtraditional freeze thaw methods.Figure 4. Consistent recovery of equally high genomic titers of AAV2 Up and AAV2 Down viral particles. AAV2 Upand AAV2 Down viral particles encoding a sgRNA targeting CCR5 and truncated portions of Cas9 were extracted fromHEK 293T cells using the AAVpro Extraction Solution. Genomic titer was measured using the AAVpro Titration Kit (forReal Time PCR). Equivalent and high genomic titers were obtained for AAV2 Up and AAV2 Down viral particles in threeindependent experiments.AAV2 delivery of Cas9 and sgRNA results in more indels compared to plasmid transfectionThe CCR5 gene, which encodes a cell surface chemokine receptor, was targeted for CRISPR/Cas9 editing.Oligos containing a guide sequence targeting the CCR5 gene were designed, annealed, and ligated to the pAAV Guide it Down plasmid. AAV2 Up and AAV2 Down viruses were packaged following the recommended protocol.HEK 293, HepG2, and MCF7 cells were then co transduced with equal number of AAV2 Up and AAV2 Downviruses. In this experiment, HEK 293 is considered an easy to transfect cell line and HepG2 and MCF7 areconsidered hard to transfect cell lines. As a control, cells were transfected with a plasmid encoding Cas9 and asgRNA targeting CCR5 by transfection.Seventy two hours after either transfection or transduction, cells were harvested and analyzed using the Guide it Mutation Detection Kit, a method that employs a mismatch specific endonuclease to identify insertions ordeletions (indels). The resulting cleavage reaction was analyzed by agarose gel electrophoresis (Figure 5). The
percentage of indels was quantified using densitometry (Cong et al., 2013).As anticipated, plasmid transfection of Cas9 sgRNA yielded significant indel formation only in HEK 293 cells.For the hard to transfect cells, HepG2 and MCF7 cells, there was almost no detectable indel formation (Figure5).Conversely, AAV2 mediated delivery of Cas9 sgRNA yielded significant indel formation in both HEK 293 andthe hard to transfect cells (Figure 5). Interestingly, the fold difference in indel formation between transfectionand transduction was far more pronounced in HepG2 and MCF7 cells than HEK 293 cells (5.6 and 6.3 foldversus 2.0 fold). These data underscore the efficacy of the AAVpro CRISPR/Cas9 Helper Free System (AAV2)at inducing site specific genomic modifications in hard to transfect cells.Figure 5. The AAVpro CRISPR/Cas9 system results in more indel formation than plasmid transfection especially inhard to transfect cell lines. The indicated cell types (1.0 x 105 cells) were seeded in 12 well plates one day beforeeither transfection or transduction. Cells were transduced with 1.0 x 105 MOI (genomic titer) each of AAV2 Up andAAV2 Down viral particles targeting the CCR5 gene. Genomic titers were calculated using the AAVpro Titration Kit (forReal Time PCR). After 72 hours, cells were harvested and analyzed using the Guide it Mutation Detection Kit. As acontrol, Guide it ZsGreen1 plasmid (P: 2.5 µg) targeting the CCR5 gene was introduced by transfection. As a negativecontrol (" "), cells without either transfection or transduction were included in the analysis. Indel formation (%) wasassessed by agarose gel electrophoresis and measured using densitometry.ConclusionsThe AAVpro CRISPR/Cas9 Helper Free System (AAV2) is a complete system for the delivery of Cas9 andsgRNA to mammalian cells using AAV2. The kit contains all of the necessary reagents to prepare infection ready, high titer AAV particles to deliver Cas9 and a user defined, gene specific sgRNA. AAV mediated deliveryof Cas9 and sgRNA using this system, results in greater genome editing compared to plasmid transfectiondelivery, especially in hard to transfect cells.ReferencesByrne, S.M. et al. (2014). Genome editing in human stem cells. Methods in Enzymology 546:119–138.Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas9 systems. Science339(6121):819–23.Hirsch, M. et al. (2010) Little Vector, Big Gene Transduction: Fragmented genome assembly of adeno associated virus. Molecular Therapy nome Editing/CRISPR Cas9/Technical Notes/CRISPR-Cas9 editing in hardto-transfect cells
Improved CRISPR/Cas9 Genome Editing in Hard to Transfect Mammalian Cells Using AAV AAV mediated delivery of Cas9 and sgRNA expression cassettes results in more indels, especially in hard to transfect cell lines: . The use of CRISPR/Cas9 technology can be limited by delivery options for Cas9 and the single guide RNA (sgRNA). .
nents comprising CRISPR genome editing, each of which is considered next. Components of CRISPR Genome Editing Component 1: Cas9 Endonuclease The most common endonuclease used in CRISPR genome editing is the class II effector protein, Cas9, from S pyogenes (
Adding CRISPR to your Bio ARROW Protocol Page 2 of 6. Work Covered by this Guidance Document: This guidance document covers how to add the use of CRISPR systems (e.g., CRISPR/Cas9, CRISPR/Cpf1) – whether for genome editing or other purposes (e.g., CRISPR-mediated
Cas9 Enzymology The Cas9 protein contains two independent endonuclease domains: one is homologous to the HNH endonuclease . CRISPR/Cas9 Delivery Methods tool, CRISPR was widely used in many experimental settings . RNPs induce editing at. 3 .
of gene targeting” . Thus, the CRISPR-Cas9 system is poised to transform genome editing. CRISPR-Cas9 technology is derived from a bacterial adaptive immune system. It is a two-component system that depends on an enzyme (Cas9) to cleave double-stranded DNA, and a guide RNA (gRNA) that
1.3 The development of CRISPR/Cas genome-editing technology 380 1.4 The zebrafish animal model and CRISPR/Cas 383 2. Targeted Generation of Indel Mutations 385 2.1 Cas9 modification and delivery platforms 385 2.2 Single-guide RNA design considerations 388 2.3 Introduction and identification of Cas9-sgRNA-induced indels 395 3.
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes Chen et al., JBC, 2016 CRISPR-EZ Advantages 100% Cas9 RNP delivery Highly efficient NHEJ and HDR editing indel, point mutation, deletion, insertion 3x increase in embryo viability Easy, economic and high-throughput CRISPR-EZ Challenges Larg
development via CRISPR-Cas9 and synthetic biology technologies 2. CRISPR/Cas9 gene editing of Theileria parva for the development of vaccine against East Coast fever (ECF) T b Specifics of the target gene(s) and phenotype(s): Animals: pigs (African Swine Fever Virus) and cattle (Theileria parva) PROJECT TITLE: KENYA O p 13 Dr. Hussein Abkallo
and sgRNA sequences in the paper model match those used in Activity 2, lacZ CRISPR Gene Editing laboratory. 1. Cut out the sgRNAs and DNA strips. You may leave the Cas9 protein on its page. 2. Use the steps in Figure 2 as a guide to model the CRISPR-Cas9 mechanism: a. Cas9 binds an sgRNA:
Make sure your target sequence is what you think www.ensembl.org (and sequence verify) Design your guide (GG-18N-NGG) crispr.mit.edu/ chopchop.rc.fas.harvard.edu/ Clone your guide into proper Cas9 exp
initial heterozygous mutant. In mammalian cell culture, . using CRISPR–Cas9 Ophir Shalem, Neville E. Sanjana and Feng Zhang . genes and is not necessarily required for Cas9 -mediated knockout, as an early frameshift mutation or large indels might be sufficient to produce a non-functio
Congressional Research Service R44824 · VERSION 5 · UPDATED 3 base9 in a gene (base editing), cut a single strand of DNA, or activate or repress the expression of a gene (i.e., increase or decrease the production of a molecule, typically a protein).10 What Are Gene Drives? CRISPR-Cas9 has led to recent breakthroughs in gene drive research.
CRISPR ethics and vice versa. Third, we assess several key ethical considerations. Notably, while some of these concerns are specific to CRISPR technology, many, such as research on human embryos, have been debated long before the CRISPR revolution . Moreover, since CRISPR is still a maturing technology, novel applications in
alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain. Keywords: CRISPR, Cas9, Knock-in mice, Rosa26, Zygotes Background The Rosa26 locus on chromosome 6 is frequently used for the integration of transgene constructs to achie
mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applicationsfor generating targetedgenome modifications and genereg-ulations in plants. Methods in Enzymology, Volume 546 # 2014 Elsevier Inc. ISSN 0076-6879 All rights .
The human genome is the first genome entirely sequenced. b. The human genome is about the same size as the genome of E. coli. c. Researchers completed the genomes of yeast and fruit flies during the same time they sequenced the human genome. d. The sequence of the human genome was completed in June 2000. 10.
Development of CRISPR-Cas systems for genome editing and beyond F. Zhang 1Broad Institute of MIT and Harvard, Cambridge, MA 02142 USA; 2Department of Brain and Cognitive Sciences, Department of Biological Engineering, McGovern Institute for Brain Research, Massachusetts Institute of
based methods for target validation, drug-target pairs can be identified and validated in model systems, such as mammalian cells, as well as model organisms. NEB provides reagents to support a broad variety of CRISPR/Cas9 genome editing approaches. From the introduction of Cas9 and single guide RNA (sgRNA) into
Sigma-Aldrich, the industry leader in genome editing, presents CRISPR Synthetic Guide RNA to fast track your research. Target any sequence in any species with Sigma crRNA. The Sigma two-part gRNA system is ready-to-use with Cas9 protein or Cas9 mRNA and compatible with a variety of delivery methods such as microinjection,
Figure 1: CRISPR/Cas9-setup. (A) Cas9-gRNA-mediated in vitro cleavage of the Ltv1 DNA with gRNAs targeting exons 1, 2 and 3 of Ltv1. (B) T7 endonuclease assay after Cas9/gRNA transfection shows cleavage, indicating successful ge
matched to the Cambridge IGCSE and O Level Accounting syllabuses, this coursebook increases understanding of accounting best practice. Clear step-by-step explanations and instructions help students learn how to record, report, present and interpret nancial information while gaining an appreciation of the ways accounting is used in modern business contexts. The coursebook is ideal for those .