CAS9 AS A VERSATILE TOOL FOR ENGINEERING BIOLOGY PDF

The speed and ease of implementing these technologies has led to an explosion of mutant and transgenic organisms. CHOPCHOP accepts a wide range of inputs gene identifiers, genomic regions or pasted sequences and provides an array of advanced options for target selection. Each query produces an interactive visualization of the gene with candidate target sites displayed at their genomic positions and color-coded according to quality scores. In addition, for each possible target site, restriction sites and primer candidates are visualized, facilitating a streamlined pipeline of mutant generation and validation. TALENs are a genome editing method derived from plant pathogenic bacteria 2. Importantly, the RVDs can be assembled sequentially to bind any given target sequence.

Author:Mauzil Mokree
Country:Peru
Language:English (Spanish)
Genre:Literature
Published (Last):3 September 2018
Pages:375
PDF File Size:7.42 Mb
ePub File Size:14.48 Mb
ISBN:743-6-46807-196-9
Downloads:89758
Price:Free* [*Free Regsitration Required]
Uploader:Kazrazuru



Author manuscript; available in PMC June Published in final edited form as: Nat Methods. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.

Type II CRISPR-Cas systems have been engineered to effect robust RNA-guided genome modifications in multiple eukaryotic systems4—17, substantially improving the ease of genome editing and, more recently, genome regulation18— As an RNA-guided dsDNAbinding protein, the Cas9 effector nuclease is the first known example of a programmable unifying factor capable of colocalizing all three types of sequence-defined biological polymers, a capability with tremendous potential for engineering living systems.

Here we review the Cas9 targeting methodology, outline key steps toward enhancing the efficacy, specificity and versatility of Cas9-mediated genome editing and regulation, and highlight its transformative potential for basic science, cellular engineering and therapeutics.

Transcription of the locus yields a pre-crRNA, which is processed to yield crRNAs consisting of spacer-repeat fragments that guide effector nuclease complexes to cleave?

All rights reserved. Correspondence should be addressed to P. Mali et al. Page 2 dsDNA sequences complementary to the spacer. Third, each mature complex locates a target dsDNA sequence and cuts both strands. Implementing this system in a given organism requires appropriate reconstitution of the functional crRNA-tracrRNA-Cas9 functional unit. In bacteria, the system can be used as is28, but in the human setting this involves expression of a human-codon—optimized Cas9 protein with an appropriate nuclear localization signal, and the crRNA and tracrRNA expressed either individually or as a single chimera via a RNA polymerase III promoter5,11, Alternatively, in vitro— transcribed RNA can be delivered directly to the target cell types4, This general methodology has been used to edit genomes of numerous model eukaryotic organisms4— Expanding Cas9 functionality For all its demonstrated utility in facilitating genome editing, we suggest that the true versatility and potential of the Cas9 unifying factor is in its singular ability to bring together all three major classes of biopolymers.

Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to Cas9nuclease-null or to sgRNAs, or by noncovalent binding to covalently attached molecules Fig. Because so many biological elements are primarily regulated by effective concentration, a single unifying Nat Methods.

Page 3 factor capable of mediating these interactions has extraordinary potential for use in investigating and engineering living systems Fig. By targeting Cas9nuclease-null to important binding sites for putative transcription factors, it should be possible to obstruct the recruitment of these factors and thereby elucidate their role in transcription. Similarly, individual factors with unknown roles could be selectively recruited to almost any desired sequence by Cas9nuclease-null fusions or sgRNA tethers with only slightly less precision.

Together, these capabilities may allow a reductionist, component-by-component approach to perturbing endogenous gene regulation. Transcriptional activation For engineering purposes, it is most useful to directly upregulate the transcription of endogenous genes to a desired level of activity. We and others showed that Cas9-mediated localization functions similarly with both Cas9nuclease-null—VP64 refs.

As a caveat, the extent of activation can vary markedly among targeted genes and requires synergy between multiple Cas9-sgRNA activators for robust transcription, presumably owing to local chromatin structure, unique interactions with endogenous transcriptional machinery or the underlying Cas9 biochemistry.

Elucidation of these effects as well as evaluation of additional Cas9 orthologs will be necessary to fine-tune our control over endogenous transcription. The capability to upregulate any endogenous gene or combination of genes in trans has tremendous implications for our ability to investigate and control cellular behavior.

In particular, multiplexed sgRNA libraries15 targeting every known gene could help pinpoint the factors responsible for important cellular processes such as differentiation. Transcriptional repression Recruitment of repressor domains by zinc finger effector or TAL effector proteins potently suppresses endogenous transcription.

By using a similar architecture for Cas9nuclease-null— KRAB or related fusion proteins or sgRNA-based tethers, it should be possible to repress genes with equivalent efficacy and far greater ease of targeting. Localizing additional repressors and optimizing the structure of the fusion protein could greatly increase the potency of this approach. Adding the ability to repress transcription to our tool-box will not only complement studies using transcriptional activation, but may also be useful for antiviral applications in eukaryotic cells.

By preventing the transcription of invading viral genomes, Cas9 repressors could in principle render a transgenic organism immune to many DNA viruses targeted with sufficient sgRNAs, a notable advantage for both crops and domesticated animals. Nat Methods. Page 4 Modulation of epigenetic marks Although no attempts to engineer chromatin modifications at endogenous loci using Cas9 have been published, recruiting the appropriate effector domains should result in the desired effects.

In principle, Cas9 can precisely recruit any of the major chromatin-remodeling complexes, including Swi-Snf, histone acetylases and deacetylases, methylases and demethylases, kinases and phosphatases, DNA methylases and demethylases, and others. Modulation of genome architecture Regulation may also be achieved via programmable alterations to genome architecture41, Cas9nuclease-null has the potential to bring together any two or more regions of the genome via multivalent sgRNAs that recruit Cas9 and also other sgRNAs.

Alternatively, a single sgRNA with multiple Cas9nuclease-null binding sites and spacers might have a similar role in bringing together two target dsDNA regions. These genomic rearrangements might in turn be visualized by attaching fluorescent proteins to the Cas9nuclease-null—effector complex by direct fusion, sgRNA tethering or both.

Cas9-targeted recombinases Despite the effectiveness of nuclease-based methods in editing genomes, safe in vivo gene correction in human patients remains difficult. Most notably, the introduction of a doublestrand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may culminate in oncogenesis.

Site-specific recombinase and potentially transposase enzymes present fewer problems by tightly controlling generation of double-strand breaks to coordinate donor-target coupling. By fusing the catalytic domain of a small serine recombinase44 to Cas9, analogous to previous zinc finger and TAL fusions45, it may be possible to create an RNA-guided recombinase enzyme. Because the activity of such retargeted fusion recombinases is generally low, extensive directed evolution may be necessary to produce a useful RNA-guided recombinase.

These are by nature constitutive promoters, and transcribed RNAs have limited total lengths and short half-lives. Successful expression of sgRNAs using polymerase II promoters could enable coordinated and inducible control over multiple aspects of cellular behavior as well as production of multiple sgRNAs from a single transcript. Unfortunately, most polymerase II transcripts are rapidly exported to the cytoplasm.

Tightly controlling the dose and duration of Cas9-sgRNA expression will also be critical for tuning targeting specificity. If one of the two nuclease domains is inactivated, Cas9 will function as a nickase Both modalities have been demonstrated to induce nonhomologous end joining NHEJ -mediated disruption of the genome and homologous recombination HR -mediated modification of the genome in eukaryotic systems5,15, However, the intricacies of this process remain poorly understood, as exemplified by the wide range of efficiencies and specificities observed in many systems.

Determining targeting biases Spacer sequences vary dramatically in their targeting efficiencies in both eukaryotic and prokaryotic systems, and PAM sequences also have a role in targeting For example, a spacer targeting the unc locus yielded gene disruptions in only 1.

In eukaryotes, we observed the frequency of insertions versus deletions during NHEJ to vary substantially between target sites Similar confounding aspects were also found when working with nickase versions of Cas9. In particular, use of the nickase did not result in any detectable NHEJ at a large majority of target sites5,21,52 but induced a very high rate at certain target sequences for unknown reasons More generally, the extent to which underlying chromatin structure, DNA modifications or cell type—specific contributions affect Cas9-sgRNA targeting remains unknown.

Rigorous quantification of all these influences is urgently needed to construct predictive models of Cas9 targeting, especially as we begin to design large sgRNA libraries for genetic screens. Improving specificity An increasingly recognized constraint limiting Cas9-mediated genome engineering applications concerns their specificity of targeting.

The sgRNA-Cas9 complexes are in general tolerant of 1—3 mismatches in their target and occasionally more, with the actual specificity being a function of the Cas9 ortholog, the sgRNA architecture, the targeted sequence, the PAM, and also the relative dose and duration of these reagents21,52— Although imperfect Cas9 specificity is a major reason for concern, there are several methods of potentially improving this.

Broadly, these include requiring multiple sgRNA-Cas9 complexes for activity21, reducing affinity while increasing cooperativity, establishing competition between inactive and active forms, discovering improved natural orthologs55, engineering improved variants and judiciously choosing targeting sgRNAs5,15,21, Page 6 Requiring cooperativity Obligate Cas9 cooperativity, i. The most straightforward option for genome-editing purposes is to employ nickase enzymes rather than nucleases21,56— Two offset nicking events can be used to create a double-strand break with a defined overhang rather than using a single nucleolytic event to produce a blunt cut Because a majority of nicks do not result in NHEJ events, only the coordinate nicks at the targeted site will initiate a genome editing event.

By tailoring the overhangs generated, this approach can potentially be used to steer the genome repair machinery toward HR or NHEJ as desired. Another route to improving specificity would couple dimerization-dependent nuclease domains, such as FokI, to a nuclease-null Cas9, thereby requiring coordinate binding by two adjacent Cas9-sgRNA complexes to dimerize otherwise weakly interacting FokI monomers and hence effect double-strand breaks Discovering or evolving improved Cas9 proteins It is possible that certain Cas9 orthologs might prove more specific than the Cas9 from S.

The specificity of natural Cas9 proteins is likely determined by the evolutionary fitness cost of genome cutting by new and existing spacers. Consequently, it is unlikely that Cas9 proteins with longer PAM requirements will exhibit greater overall specificity, as the net selective pressure for accurate recognition of the combined spacer and PAM remains constant.

However, Cas9 proteins from species with larger genomes may be somewhat more specific, and those that have undergone frequent horizontal gene transfer along with their CRISPR loci and consequently been selected for avoidance of multiple host genomes are likely the most specific of all.

The best Cas9 proteins identified in nature might be improved by rational design, directed evolution or ideally a combination of the two. Our ability to rationally modify the enzyme is presently constrained by the lack of a crystal structure, a deficit that must be remedied. Alternatively, the PAM might be changed to expand the range of targetable sites or enlarged to increase specificity, although such alteration may not be accessible by rational design alone.

PAM alteration and more complex modifications might be accessible using directed evolution, including increasing the overall specificity of each Cas9 monomer. Such an experiment must be designed to select for activity at a perfectly matched protospacer and against activity at mismatched sites, preferably those identified as problematic by specificity measurement assays.

Ideally, the process would result in selection against many mismatched protospacers at any one time, and the process would be repeated over many rounds of selection, rendering automated methods of directed evolution particularly well-suited to this challenge Page 7 Target-site selection Judiciously choosing the targeting sgRNAs themselves will also be critical to achieving highly specific modifications.

Moreover, we and others observed dramatic differences in the extent to which different spacers tolerate mismatches, suggesting that multiple candidate spacers should be empirically tested for applications that require great specificity. Ideally, these differences would be predicted computationally, but additional experiments are needed to elucidate the rules governing spacer-dependent specificity21,52— One can target multiple genes simultaneously and also harness synergies between multiple activators or repressors.

In principle, array-based oligonucleotide synthesis15,63 could be used to produce nearly designed sgRNAs at once, a library capable of targeting every gene in the human genome 20 times. Careful multistep methods for creating libraries could allow each synthesized oligonucleotide to encode multiple sgRNAs for synergistic regulation.

Combined with appropriate screening methodologies, these library-based approaches could identify individual genes or even combinations that regulate a variety of phenotypes in eukaryotic organisms and cells Fig. Coupled with tools that enable multiplexed monitoring of the resulting changes64—66, this multiplexing ability of the sgRNA-Cas9 system15 will have potentially far-ranging implications for our ability to understand and control the factors governing cellular differentiation and behavior.

Given the abundance of potential Cas9-mediated functions, it will be necessary to develop methods for independent targeting such that each function exclusively responds to its own set of guide RNAs. By carefully choosing and characterizing Cas9 orthologs with widely disparate repeat sequences, it is possible to identify fully orthogonal sets of sgRNA-Cas9 pairings and hence to simultaneously execute multiple functionalities by engineering each ortholog with a custom effector domain For example, three orthogonal Cas9 proteins would allow activation, repression and editing to be performed simultaneously at independent target sites in the same cell.

Toward Cas9 therapeutics Given the tremendous utility of Cas9 for the regulation and modification of complex biological systems, might the Cas9 system prove equally useful as a basis for new therapeutics?

We envision two primary routes to Cas9-mediated therapeutic interventions Nat Methods. Page 8 Fig. The first entails targeted genome editing to correct genetic disorders68—70 and possibly to disrupt invading viral genomes.

The second will use Cas9nuclease-null fusions for targeted genome regulation in a manner akin to the use of small-molecule drugs, except that both repression and activation modalities would be available. One can imagine using such an approach to correct epi-genetic misregulation of gene expression, to control inflammation and autoimmunity, and also to repress transcription of viral genes or even viral co-receptors in vulnerable cell types.

However, multiple technical hurdles must be addressed before Cas9-based therapeutics become a reality.

ELITEPAD 900 SPECS PDF

Cas9 as a versatile tool for engineering biology.

Author manuscript; available in PMC June Published in final edited form as: Nat Methods. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic. Type II CRISPR-Cas systems have been engineered to effect robust RNA-guided genome modifications in multiple eukaryotic systems4—17, substantially improving the ease of genome editing and, more recently, genome regulation18— As an RNA-guided dsDNAbinding protein, the Cas9 effector nuclease is the first known example of a programmable unifying factor capable of colocalizing all three types of sequence-defined biological polymers, a capability with tremendous potential for engineering living systems.

DIPLOMAT ADP8332 MANUAL PDF

Cas9 as a versatile tool for engineering biology

Monos Avoiding an adverse immune response is also critical. Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to Cas9 nuclease-null or to sgRNAs, or by noncovalent binding to covalently attached molecules Fig. Cas9 as a versatile tool for engineering biology Zhang F, et al. Combined with appropriate screening methodologies, these library-based approaches could identify individual genes or even combinations that regulate a variety of phenotypes in eukaryotic organisms and cells Fig. Most notably, the introduction of a double-strand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may culminate in oncogenesis. Zhang Y, et al. Together, these capabilities may allow a reductionist, component-by-component approach to perturbing endogenous gene regulation.

FARMACOS ANTIARRITMICOS PDF

CAS9 AS A VERSATILE TOOL FOR ENGINEERING BIOLOGY PDF

.

PSYCHOLOGIA DZIECKA SCHAFFER PDF

.

Related Articles