Name: a universal protocol for large-scale grna library production from any dna source
Uploaded: 2017-12-06T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Universal Protocol for Large-scale gRNA Library Production from any DNA Source at JoVE.com

The authors would like to thank Prof. Dr. Stephan Beck and Prof. Dr. Magdalena Goetz for their input, help and support in developing the CORALINA method, Maximilian Wiessbeck and Valentin Baumann for helpful comments. The work has been supported by DFG (STR 1385/1-1).

<ol>
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The authors have nothing to disclose.

CORALINA can be used to generate large scale gRNA libraries by controlled nuclease digestion of target DNA and bulk cloning of resulting double stranded fragments. Statistical inference indicates that many more than 107 individual gRNA sequences have already been successfully cloned using the protocol at hand9. CORALINA can be customized in multiple ways. The choice of template DNA defines the target region and the maximal complexity of the generated library. Using this protocol, CORALINA libraries have previously been generated from human and mouse genomic DNA9. Representative results presented here depict the generation of a CORALINA library from purified BAC DNA. Further customization can be achieved by the choice of gRNA expression vector and linker sequences. We have previously tested three different pairs of linker lengths for Gibson assembly with little variations in efficiency9.
Due to their origin from bulk digested DNA, protospacer of CORALINA gRNAs are usually not exactly 20 bp in length, but show a length distribution with a mean that depends both on the parameters of the MNase digestion as well as the size of the excision made from the PAGE gels. The representative example shown in Figure 2B and C, depicts fragments with a median length between 19 and 27 bp. In our experience, the length of the fragments is faithfully preserved by the generated gRNA protospacer9. While fragments shorter than 20 bp should be avoided due to higher off-target rate of resulting gRNAs, longer fragments are likely much less of a problem for downstream applications, since it has been demonstrated that gRNAs with protospacers as long as 45 bp are still functional9.
The two most critical steps in the CORALINA protocol are the size selection of MNase-digested fragments and cloning steps. Generation of fragments that are too short (e.g. average below 18 bp) or incorporation of too many empty gRNA expression vectors will render the library useless. Thus, it is important to optimize the MNase digestion step (Figure 2A), to monitor excision (Figure 2B, C), check for complete digestion of the gRNA vector backbone and including no fragment controls throughout the protocol. Special care has also to be taken to preserve the representation of the gRNA library. One common bottleneck of library generation in general is the efficient transfer of plasmids into bacteria for amplification. Thus, large quantities of bacteria with excellent competency and a large number of individual electroporation events are necessary to achieve a high number of gRNA clones.
New strategies for gRNA library production will be necessary to harvest the full potential of CRISPR-based screening approaches over the next decades. There is a significant demand for cost-effective, simple and customizable methods to generate large-scale libraries, a pre-requisite to make screening amenable to a larger number of model systems and different CRISPR-based engineering approaches. CORALINA is providing a first step toward this. The potential uses are manifold, especially to produce comprehensive libraries of genomes, cDNA derived libraries of less common model systems, highly focused libraries and experimental set-ups in which different CRISPR proteins (with differing PAM requirements) are used in combination.
Unlike other methods, CORALINA generates all possible gRNAs from the input DNA. However, one drawback of the method is that gRNAs lacking the required PAM sequence are also included in the library, a feature that it shares with a second enzymatic method for gRNA library generation, CRISPR-EATING (Table 1). The choice of the ideal method for gRNA library generation depends on the specifications of the planned screening experiment, especially the nature (genic, regulatory, intergenic) and size of the target region (single locus, multiple regions, genome-wide). We see a special upside in using CORALINA when a large number of non-coding or regulatory regions are to be analyzed, if there is incomplete or unreliable sequence information (exotic model systems, mixtures of species (e.g. microbiomes) or experimentally obtained input), if different CRISPR endonucleases are combined or if saturating analysis is performed on a short and defined locus (e.g. represented by BACs).

The adaptation of the bacterial CRISPR/Cas9 system as a molecular targeting tool caused the most recent revolution in molecular biology. Never before has it been so easy to manipulate chromatin at defined genomic locations. Common applications of CRISPR include targeted gene mutations1, genome engineering2, epigenome editing3, transcriptional activation and gene silencing4. One particular advantage of the CRISPR system is that its applications are not limited to well-studied candidate sites, as gRNA libraries make less biased screens possible. These facilitate the discovery of functional loci in the genome without any prior experimental knowledge. However, gRNA library construction is currently mostly based on oligo-nucleotide synthesis, and there are limited options to purchase gRNA libraries that are not of human or mouse origin or target regions outside open reading frames. Thus, although CRISPR screens have already proven incredibly potent5,6,7,8, their full potential has not yet been exploited.
To overcome the limitation of classical gRNA generation methods two strategies have recently been developed. Both are based on controlled enzymatic digestion of target DNA rather than relying on custom oligonucleotide synthesis. While CORALINA9 employs micrococcal nuclease, the only currently available alternative method, CRISPR-EATING10, makes use of restriction enzymes (HpaII, ScrFI, BfaI and MmeI). Importantly, both techniques can be applied to any input DNA, which serves as the source of gRNA protospacer sequences. While the CRISPR-EATING method employs a strategy to decrease the number of cloned gRNAs whose targeting sites are not followed by the required S.pyogenes PAM (protospacer adjacent motive), it generates only a small fraction of all possible functional gRNAs for a given region. CORALINA, on the other hand, is able to generate all potential gRNAs for the source sequence, but also incorporates a higher fraction of non-functional guides. gRNA library generation through controlled nuclease activity enables the production of comprehensive gRNA libraries for any species, any Cas9-protein or -effector system in a simple and cost-effective manner. Moreover, CORALINA is adaptable to customization, as appropriate input and vector choices define the library type, size and content. Here, a detailed protocol is presented that can be used for the generation of comprehensive gRNA libraries from diverse sources of DNA (Figure 1), including bacterial artificial chromosomes (BACs) or genomic DNA9. The representative results accompanying this protocol were derived by applying the CORALINA protocol to BAC DNA.

<table><tbody><tr><td>500 mM EGTA</td><td>Sigma Aldrich</td><td>03777-10G</td><td>1.4., Inactivation of Mnase</td></tr><tr><td>Novex Hi-Density TBE Sample Buffer</td><td>Thermo Fisher Scientific</td><td>LC6678</td><td>2.1.</td></tr><tr><td>Novex&amp;reg; TBE Gels, 20%, 10 well</td><td>Thermo Fisher Scientific</td><td>EC6315BOX</td><td>2.1., pre-made 20 % PAGE gel</td></tr><tr><td>O&#039;RangeRuler 5 bp DNA Ladder,</td><td>Thermo Fisher Scientific</td><td>SM1303</td><td>2.1.</td></tr><tr><td>Novex&amp;reg; TBE Running Buffer</td><td>Thermo Fisher Scientific</td><td>LC6675</td><td>2.1., PAGE gel running buffer</td></tr><tr><td>Disposable scalpel, sterile</td><td>VWR&amp;nbsp;</td><td>233-5363&amp;nbsp;</td><td>2.3., other equivalent reagents may be used</td></tr><tr><td>SYBR Green I nucleic acid stain (1000x concentrate in DMSO)</td><td>Sigma Aldrich</td><td>&lt;br/&gt; S9430</td><td>2.3. +2.5., also available from Thermo Fisher Scientific (S7563)</td></tr><tr><td>UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td><td>Thermo Fisher Scientific</td><td>15593-031</td><td>3.6.1. + 4.3., other equivalent reagents may be used</td></tr><tr><td>Glycogen</td><td>Sigma</td><td>10901393001</td><td>3.6.4., other equivalent reagents may be used</td></tr><tr><td>3M Sodium acetate , pH5.2</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp; R1181</td><td>3.6.4., other equivalent reagents may be used</td></tr><tr><td>Ethanol&amp;nbsp;</td><td></td><td></td><td>3.6.4. + 9.1.8., molecular biology grade</td></tr><tr><td>Quick blunting kit&amp;nbsp;</td><td>New England Biolabs</td><td>E1201</td><td>4.1.</td></tr><tr><td>ammomium acetate</td><td>Sigma</td><td>&lt;br/&gt; A1542</td><td>3.1., other equivalent reagents may be used</td></tr><tr><td>magnesium acetate</td><td>Sigma</td><td>&lt;br/&gt; M5661</td><td>3.1., other equivalent reagents may be used</td></tr><tr><td>0.5 M EDTA (pH 8.0)</td><td>VWR</td><td>&amp;nbsp; MOLEM37465520 (or Promega V4231)</td><td>2.2. + 3.1., other equivalent reagents may be used</td></tr><tr><td>Agencourt AMPure XP beads</td><td>Beckman coulter</td><td>A63881</td><td>5.3. + 6.5.</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>5.7.+ 7.1. +8.4., other equivalent reagents may be used</td></tr><tr><td>concentrated T4 DNA ligase</td><td>New England Biolabs</td><td>&amp;nbsp;M0202T</td><td>6.1.+ 8.1.2.</td></tr><tr><td>Long Amp Taq 2X Master Mix&amp;nbsp;</td><td>New England Biolabs</td><td>M0287S</td><td>6.3.</td></tr><tr><td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td><td>New England Biolabs</td><td>M0531S</td><td>5.1. + 6.6., other equivalent reagents may be used</td></tr><tr><td>HindIII</td><td>New England Biolabs</td><td>R0104S</td><td>5.4.1.&amp;nbsp;</td></tr><tr><td>SacII</td><td>New England Biolabs</td><td>R0157S</td><td>5.4.2.</td></tr><tr><td>AgeI</td><td>New England Biolabs</td><td>R0552S</td><td>8.2.1.</td></tr><tr><td>Tris base</td><td>Sigma</td><td>93362</td><td>8.1.1.</td></tr><tr><td>2M MgCl</td><td>Sigma</td><td>93362</td><td>8.1.1.</td></tr><tr><td>dGTP,dATP, dCTP, dTTP</td><td>New England Biolabs</td><td>N0446S</td><td>8.1.1.</td></tr><tr><td>DTT</td><td>Sigam</td><td>&lt;br/&gt; DTT-RO</td><td>8.1.1.</td></tr><tr><td>PEG-8000&amp;nbsp;</td><td>Sigma</td><td>&lt;br/&gt; P5413</td><td>8.1.1.</td></tr><tr><td>NAD</td><td>Sigma</td><td>&lt;br/&gt; N6522</td><td>8.1.1.</td></tr><tr><td>T5 exonuclease</td><td>New England Biolabs</td><td>M0363S</td><td>8.1.2.</td></tr><tr><td>Phusion DNA polymerase&amp;nbsp;</td><td>New England Biolabs</td><td>M0530S</td><td>8.1.2.</td></tr><tr><td>Taq DNA ligase</td><td>New England Biolabs</td><td>M0208L</td><td>8.1.2.</td></tr><tr><td>rSAP</td><td>New England Biolabs</td><td>M0371S</td><td>8.3.1.</td></tr><tr><td>TG1 competent cells</td><td>Lucigen</td><td>60502-1</td><td>9.1.</td></tr><tr><td>1mm gap electroporation cuvettes&amp;nbsp;</td><td>VWR</td><td>732-2267&amp;nbsp;</td><td>10.2.</td></tr><tr><td>Bio-Assay Dish (Polystyrene, 245 mm x 245 mm x 25 mm)</td><td>Fisher Scientific</td><td>DIS-988-010M&amp;nbsp;</td><td>9.4.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653&amp;nbsp;</td><td>9.3.</td></tr><tr><td>Bacto-tryptone</td><td>BD</td><td>211705</td><td>9.3.</td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td>9.3.</td></tr><tr><td>Agar</td><td>Sigma</td><td>&lt;br/&gt; A1296</td><td>9.4.</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>&lt;br/&gt; G5516</td><td>9.17.</td></tr><tr><td>MNAse</td><td>New England Biolabs</td><td>M0247S</td><td>1.1.</td></tr><tr><td>Nanodrop</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td>throughout</td></tr><tr><td>Micropulser</td><td>Biorad</td><td>165-2100</td><td>10.2.</td></tr><tr><td>Electroporation cuvettes</td><td>Biorad</td><td>732-2267&amp;nbsp;</td><td>10.2.</td></tr><tr><td>250 ml centrifuge tubes</td><td>Corning</td><td>430776</td><td>9.1-9.9.</td></tr></tbody></table>

a universal protocol for large-scale grna library production from any dna source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Using the protocol at hand, CORALINA gRNA libraries have been generated from human and mouse genomic DNA9 and BAC DNA (Figure 1). To produce fragments of input DNA suitable for cloning into gRNA expression vectors, optimal conditions for controlled nuclease digestion have to be determined. A typical result for the optimization of micrococcal nuclease digestion is depicted in Figure 2A. Insufficient amount of nuclease (0.1, 2, 3, 4, 4.5 or 5 units) produces no noticeable products in the required size range (10-100 bp) and 5.5-7.5 units still produced fragments that are on average too long. Larger amounts of enzyme (50 units) lead to excessive degradation of input DNA after 10 min. Consequently, an intermediate amount was chosen (10 units). The digest was scaled up to produce enough digested fragments for subsequent purification and cloning (Figure 2B). While it is recommended to blindly select DNA fragments by size and only rely on the DNA ladder for orientation to minimize exposure of DNA fragments to UV light, gels can be stained afterwards for quality control of digestion and cutting. Figure 2B shows a representative example of a PAGE gel from which DNA fragments between 20 and 30 bp have been excised. Gel purified MNase fragments were loaded onto a 20% PAGE gel to check successful size selection and purification of MNase-digested fragments (Figure 2C). The protocol at hand is compatible with the use of customized linker sequences, allowing to clone the MNase-digested fragments into gRNA expression vectors of choice. Here, gRNA-PLKO9 was used as backbone. The linkers are amplified from the gRNA expression vector using standard PCR. Figure 2D depicts a representative example of amplified linker sequences devoid of additional, incorrect or no template amplicons. Next, linker amplicons are digested with restriction enzymes to ensure linkers are ligated onto the MNase-digested fragments in the correct orientation. Figure 2D shows agarose gels of 5&#39; and 3&#39; linkers before and after digestion with HindIII and SacII respectively, indicating complete digestion of the linkers to the predicted 637 and 295 bp. The right-hand portion of the gel image documents the excision of the digested linker fragments. Following gel extraction of digested linkers, the next step in the protocol is the ligation of linkers to the end-repaired MNase-digested fragments. Because linker sequences are generated by PCR using unphosphorylated primers, self-ligation of linkers should not occur. Only the end-repaired MNase-digested DNA fragments provide the phosphate groups necessary for ligation. Following nick translation, the ligation product is amplified by PCR. In order to avoid excessive PCR amplification bias that could skew the representation of gRNA sequences in the library, amplification is limited to less than 20 cycles in total. Following PCR, the amplification products are difficult to visualize on agarose gels. Separate control PCRs with 32 cycles are therefore performed to detect the products (but are not used for library preparation). Results from this control PCR are shown in Figure 2E. This allows to optimize the ligation reactions and to ensure reactions are devoid of PCR artefacts, which sometimes occur in &#34;no fragments controls&#34; (NFC). Figure 2E shows the desired amplicon (5&#39; linker + DNA fragment + 3&#39; linker, length: 869 bp) following amplification of ligation reactions using equimolar (1:1) ratios between fragments and linker sequences.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56264/56264fig1.jpg" /> 
Figure 1: Suggested timeline for preparing a gRNA library. CORALINA offers a simple and cost-efficient strategy for the generation of comprehensive gRNA libraries from a plethora of different DNA sources from any organism. The protocol at hand can be brought to completion during one working week. Linker generation can be performed in parallel with DNA end-repair. Preparation of electrocompetent bacteria takes two days and includes an overnight growth step and should therefore be started before assembly reactions are set up. <a href="https://cloudflare.jove.com/files/ftp_upload/56264/56264fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56264/56264fig2.jpg" /> 
Figure 2: Critical steps during the protocol. (A) Controlled digestion of BAC DNA enables generation of fragments of different sizes. Shown here is the optimization of MNase digestion. Purified BAC DNA was treated with different amounts of MNase for 10 min. 10 U of MNase generate DNA fragments of the desired length (20-30 bp). (B) Size selection of fragments between 20 and 30 bp using excision from polyacrylamide gels. Purified BAC DNA was treated with 10 U of MNase for 10 min. The image was recorded following excision. (C) Quality control of gel-purified fragments. After gel-purification, 1/6th of the purified MNase fragments was loaded onto a 20% PAGE gel to check successful size selection and purification. (D) Amplification of linker sequences for assembly and restriction enzyme digestion of linkers to ensure directional cloning. 5&#39; and 3&#39; linkers were amplified and cut with HindIII and SacII, respectively. No-template controls (NTC) were included to control for PCR artefacts and DNA contamination. Left: analytical sample application; right: preparative sample application. Image was recorded after gel excision. (E) Successful ligation of linkers to DNA fragments can be analyzed using PCR with an increased number of PCR cycles (32) and controlled by performing no template controls with H2O (NTC) or using the NTC from the previous nick translation step as input (NTC NT)). It is important to include a no fragment control (NFC), which is an amplification from a ligation and nick translation reaction from which the MNase fragments were omitted. Only samples in which MNase fragments have been combined with linker DNA produce the expected amplicon (869 bp). <a href="https://cloudflare.jove.com/files/ftp_upload/56264/56264fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Universal Protocol for Large-scale gRNA Library Production from any DNA Source at JoVE.com

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

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JoVE Journal

Genetics

11.1K Views.  Ludwig-Maximilian-Universität, BioMedical Center. Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Video: A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Environment

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Biology

A High-Throughput UAS-cDNA/ORF Plasmid Library Construction Using CRISPRmass

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5&#39; end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.&#160;

Author Spotlight: Simplifying Genome-Wide Plasmid Library Construction Using CRISPRmass

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. ...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

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Chapters in this video

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

CRISPR Guide RNA Cloning for Mammalian Systems

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Large Insert Environmental Genomic Library Production

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices