Genome Engineering Using CRISPR-Cas9 System

Part of the Methods in Molecular Biology book series (MIMB, volume 1239)

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

Key words

Genome engineering Cas9 CRISPR CRISPR-Cas9 PAM Guide RNA sgRNA DNA cleavage Mutagenesis Homologous recombination Streptococcus pyogenes 

1 Introduction

1.1 General Principle of Genome Engineering with Designer Nucleases

Genome engineering is a highly desirable technology in biological research and biomedical applications, allowing us to change genomic sequences at will to control the fundamental genetic information within a cell. It has been implemented in creating model cell lines or organisms for the study of gene functions or genetics of human diseases, and in gene therapy to correct deleterious genomic changes or confer beneficial ones. Genome engineering using engineered nucleases has been one of the most designable and scalable paths to achieve precise editing of genomic sequences. The basic principle for this process could be summarized into a two-step process (Fig. 1). The first step is the introduction of targeted DNA cleavage, in the form of typically double strand breaks (DSBs) or possibly single strand nicks (SSNs), through the activity of nucleases or nickases, respectively. The second step involves DNA repair process activated by the targeted cleavage, carried out normally by the endogenous DNA damage repair machinery within the cells. In this step, two major pathways could be employed, achieving different types of genome modification. One pathway is to repair the cleaved site via nonhomologous end joining (NHEJ) pathway, which applies only to the DSBs as SSNs are thought to be unable to induce NHEJ pathway. During NHEJ-mediated repair, insertion/deletion (indels) will be engineered into the target site. The other pathway requires the artificial supply of a repair template with chosen sequence alterations into the cell in addition to the DSBs or SSNs, so that homology-directed repair (HDR) pathway would be activated. The latter pathway requires homologous recombination (HR) between chromosomal DNA and the supplied foreign DNA stimulated by the local DNA cleavage, resulting in virtually any type of desired editing through replacement of native sequence by the designed HR template.
Fig. 1

Principle for genome engineering using designer nucleases or nickases. DNA cleavage induced by designer nucleases or DNA nicks created by designer nickases can result in double strand break (DSB), single strand nick (SSN), or double nicks (DNs) (top ). These site-specific DNA cleavages can be repaired via two different pathways (bottom ). In the error-prone NHEJ pathway for DSB or DNs, the ends of the breaks are processed by endogenous DNA repair machinery and rejoined, which can result in random indels at the site of junction. The other pathway is the HDR pathway for both DSB, SSN, and DNs, where a repair template is supplied, allowing precise editing of the endogenous genome sequences. Parts of this figure are adapted from [41]

Overall, one of the most essential rate-limiting steps in this type of genome engineering technology is the targeted cleavage of endogenous genome. Hence, effort that centers on the development of better designer DNA-cleaving enzymes has been a focus of this field. In the past decades, genome editing tools based on sequence-specific nucleases and DNA-binding proteins such as zinc-finger nucleases (ZFNs) [1, 2, 3, 4], meganucleases [5], and transcription activator like effectors (TALEs) [6, 7, 8, 9, 10, 11], among others, have opened up the possibility of performing precise perturbation of genome at single-nucleotide resolution. Recently, the emergence of a new type of genome engineering technology based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a microbial adaptive immune system, has transformed the field because of its easiness to design, rapidity to implement, low-cost, and scalability in eukaryotic cells.

The CRISPR locus in microbes typically consists of a set of noncoding RNA elements and enzymes that harbors the ability to recognize and cleave foreign nucleic acids based on their sequence signature. Three known CRISPR systems, types I, II, and III, have been described so far [12, 13, 14, 15]. Among these different CRISPR systems, type II CRISPR systems usually only require a single protein, called Cas9, to perform the target cleavage. Recent years, the type II CRISPR-Cas9 systems have been studied by several groups in their native microbial domain to elucidate their molecular organizations and functions [16, 17, 18]. These studies demonstrated that Cas9 is a RNA-guided nuclease that is capable of binding to a target DNA and introduce a double strand break in a sequence-specific manner, and its specificity is determined by sequence encoded within the RNA components.

Following these breakthroughs, this new family of RNA-guided nucleases has been successfully developed into a multiplex genome engineering system that is simple to design, efficient, and cost-effective for mammalian genome editing purposes [19, 20]. Application of this system could enable introduction of different type of modifications, such as mutation, insertion, deletion, or even larger chromosomal changes, through NHEJ or HDR pathways, in many different cell types and organisms [19, 20, 21, 22, 23, 24, 25, 26, 27].

1.2 Genome Engineering with CRISPR-Cas9 System

The required components for CRISPR-Cas9 mediated DNA cleavage are the Cas9 protein and its bound RNA component that guide the Cas9 to target sequence. The RNA component consists of a RNA duplex formed by partial pairing between the CRISPR RNA (crRNA), which includes the guide sequence that bound to target DNA through Watson–Crick base-pairing to specify the site of cleavage, and the trans-activating crRNA (tracrRNA), which facilitate the maturation of crRNA and the loading of crRNA onto Cas9 (Fig. 2a) [16, 17, 18]. Our previous work demonstrated that by harnessing the well-understood CRISPR-Cas9 system from Streptococcus pyogenes SF370, we could develop a system that is sufficient for carrying out site-specific DNA cleavage in mammalian cells [19]. A simplified version of this design is achieved by fusion of the mature form of crRNA and tracrRNA into a chimeric single guide RNA (sgRNA), which demonstrated even superior efficiency compared with the split design [19, 28, 20]. Hereafter we center on this two-component design, which enhances the convenience of using the CRISPR-Cas9 system and multiplexing of genome targeting applications.
Fig. 2

Schematic of the type II CRISPR-mediated DNA double-strand break and the design of chimeric sgRNA. (a) The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two noncoding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, 30 bp each). Each spacer is typically derived from foreign genetic material (protospacer), and directs the specificity of CRISPR-mediated nucleic acid cleavage. In the target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM) whose recognition is specific to individual CRISPR systems. The Type II CRISPR system carries out targeted DNA double-strand break (DSB) in sequential steps. First, the pre-crRNA array and tracrRNA are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA and associates with Cas9 as a duplex, which mediates the processing of the pre-crRNA into mature crRNAs containing individual, truncated spacer sequences. Fig. 2 (continued) Third, the mature crRNA:tracrRNA duplex directs Cas9 to the DNA target consisting of the protospacer and the requisite PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer. (b) Design of chimeric sgRNA for genome cleavage. The SpCas9 nuclease (yellow ) binds to genomic DNA directed by the sgRNA consisting of a 20-nt guide sequence (blue ) and a chimeric RNA scaffold (red ). The guide sequence base-pairs with protospacer target (blue ), located upstream of a requisite “NGG” called protospacer adjacent motif (PAM; colored in pink ). The SpCas9 mediates a DSB around 3 bp upstream to the 5′ end of the PAM (red triangles ). Parts of this figure are adapted from [19, 41]

The guide sequence within sgRNA has a length of 20 bp and is the exact complementary sequence of the target site within the genome, sometimes referred to as “protospacer” following the convention of microbial CRISPR research (Fig. 2a). In choosing the target site, it is important to note the requirement of having the “NGG” trinucleotide motif, called protospacer adjacent motif (PAM), right next to the protospacer target on the 3′ end. The Cas9 cleavage site is located within the protospacer and positioned at around 3 bp upstream the 5′ end of the PAM, indicating a potential anchor role of the PAM for Cas9-mediated DNA cleavage (Fig. 2a, b). The PAM sequence depends on the Cas9 protein employed in the CRISPR-Cas9 system, and hence, the “NGG” PAM sequence applies specifically to the Streptococcus pyogenes Cas9 (SpCas9) discussed in this protocol.

The Cas9 enzyme typically contains two nuclease domains: the RuvC-like (N-terminal RNase H fold) domain and the HNH (McrA-like) domain, each responsible for cleavage of one strand of the duplex DNA molecule. Therefore, there are two different versions of Cas9 that can be used for genome modification, the wild type Cas9 that induces DSBs to the target site, and the nickase version of Cas9 (Cas9n) bearing a deactivating mutation in one of the two nuclease domains, which nicks the target DNA on one strand. For SpCas9 that we described in this protocol, the two derived nickases are SpCas9n (D10A) and SpCas9n (H840A), with mutation at one of the two catalytic residues of the nuclease domain, D10 and H840, respectively. Whereas the wild type SpCas9 is highly efficient in executing NHEJ- and HDR-mediated genome engineering, its off-target effects due to the induction of DSBs at mismatched targets within the genome cannot be neglected [28, 29, 30]. The NHEJ-incompetent SpCas9n, in particular in the form of a double-nicking design where a pair of sgRNAs cooperates two adjacent nickings at target genomic locus, might be superior in terms of editing accuracy as it maintains the ability to achieve comparable genome engineering capacity and efficiency while reducing the likelihood of off-target NHEJ events [31, 32]. Furthermore, if the catalytic residues within the two domains are simultaneously mutated, the deactivated non-cleaving version of SpCas9 (dSpCas9) can serve as a generic RNA-guided DNA-binding protein, priming additional applications in mammalian cells such as targeted transcriptional modulation [33, 34, 35, 36, 37]. More recently, one study showed that by shortening the guide length within the sgRNA from 20 to 17 bp, the genome targeting specificity of wild type SpCas9 protein could be improved [38]. Shortly afterwards, two independent pieces of work demonstrated the utilization of Cas9-FokI fusion protein as a designer dimer nuclease for improved Cas9 specificity [39, 40]. All these work on SpCas9-based system and earlier demonstration that orthogonal Cas9 proteins from different microbial species can be adapted for mammalian genome targeting [19], implicate the potential of optimizing the guide RNA and the Cas9 protein as valid strategies to address the off-target issue of CRISPR-Cas9 system, raising the possibility that an efficient and specific CRISPR-Cas9 system will be realized for powerful and precision genome engineering.

Here we describe a detailed protocol for applying the CRISPR-Cas9 system from Streptococcus pyogenes for genome engineering purposes in mammalian cells. The protocol includes steps arranged in order of actual experiments: selection of targets and cloning of targeting constructs (Subheading 3.1), delivery into mammalian cells (Subheading 3.2), assay for genomic cleavage (Subheading 3.3), implementation of homologous recombination using CRISPR-Cas system (Subheading 3.4), and the verification and quantification of HR efficiency (Subheading 3.5). There are still numerous challenges related to efficiency of homologous recombination, off-target effects, and multiplexed targeting before we could deploy CRISPR-Cas9 system to achieve robust, efficient, and accurate genome engineering in all type of biological systems, especially for in vivo settings. Nonetheless, the general principle and design guidelines for SpCas9 in this chapter should be relevant for these future efforts as well.

2 Materials

2.1 Molecular Biology Reagents

  1. 1.

    Backbone plasmids: pX330 (U6-chimeric guide RNA + CBh-SpCas9 backbone, Addgene ID 42230), pX335 (U6-chimeric guide RNA + CBh-SpCas9n backbone, Addgene ID 42335). These and additional backbone plasmids mentioned in the protocol are all available from Addgene, at www.addgene.org.

     
  2. 2.

    DNA oligos (Standard de-salted) for cloning of chosen guides. For oligos longer than 60 nt, the ultramer oligo (Integrated DNA Technology, Coralville, IA, USA) is recommended.

     
  3. 3.

    PCR reagents: Herculase II fusion polymerase (Agilent, Santa Clara, CA, USA). Other high-fidelity polymerases, such as Kapa HiFi (Kapa Biosystems, Wilmington, MA, USA) can also be used for PCR in this protocol.

     
  4. 4.

    Cloning enzymes: FastDigest BbsI, FastDigest AgeI, FastAP (Thermo Scientific/Fermentas, Pittsburg, PA, USA), Plasmid-Safe exonuclease (Epicentre, Madison, WI, USA), T7 DNA ligase.

     
  5. 5.

    10× T4 DNA ligase buffer.

     
  6. 6.

    10× Tango buffer (Thermo Scientific/Fermentas).

     
  7. 7.

    DTT (DL-Dithiothreitol; Cleland’s reagent).

     
  8. 8.

    10 mM Adenosine 5′-triphosphate.

     
  9. 9.

    Ultrapure water (RNAse/DNAse-free).

     
  10. 10.

    Tris-EDTA (TE) buffer.

     
  11. 11.

    E. coli competent cells. Strains such as Stbl3 (Life Technologies, Carlsbad, CA, USA) or other recombination-deficient strain is recommended to avoid recombination of repetitive elements in the plasmids.

     
  12. 12.

    Luria Broth (LB) media.

     
  13. 13.

    Ampicillin.

     
  14. 14.

    Spin miniprep kit.

     
  15. 15.

    Plasmid Midi/Maxi Kit.

     
  16. 16.

    Standard gel electrophoresis reagents and apparatus.

     

2.2 Cell Culture and Processing Reagents

  1. 1.

    Cell line: human embryonic kidney (HEK) 293FT cell line.

     
  2. 2.

    Dulbecco’s Modified Eagle’s Medium (DMEM).

     
  3. 3.

    10 % fetal bovine serum (FBS).

     
  4. 4.

    GlutaMAX solution (Life Technologies).

     
  5. 5.

    100× Pen-Strep: 100 U/μL penicillin, 100 μg/μL streptomycin.

     
  6. 6.

    OptiMEM medium (Life Technologies).

     
  7. 7.

    TrypLE™ Express Enzyme (Life Technologies).

     
  8. 8.

    Transfection reagent: Lipofectamine 2000 (Life Technologies) can be used for HEK 293FT or Neuro-2a cell lines with this protocol. Other transfection reagents can also be used depending on the cell type and after trial/optimization.

     
  9. 9.

    24-well tissue culture plates.

     
  10. 10.

    pCMV-EGFP plasmid (Addgene, Cambridge, MA, USA).

     
  11. 11.

    pUC19 plasmid (NEB).

     

2.3 Genome Modification and Homologous Recombination Assay Reagents

  1. 1.

    1× PBS.

     
  2. 2.

    QuickExtract™ DNA extraction kit (Epicentre).

     
  3. 3.

    SURVEYOR Mutation Detection Kit (Transgenomic, Omaha, NE, USA).

     
  4. 4.

    4–20 % Novex TBE polyacrylamide gels and accompanying gel-running reagents (Life Technologies).

     
  5. 5.

    PCR primer for amplification of genomic DNA used in SURVEYOR assay and HR assay.

     
  6. 6.

    PCR reagents: Herculase II fusion polymerase (Agilent). Other high-fidelity polymerases, such as Kapa HiFi (Kapa Biosystems) can also be used for PCR in this protocol.

     
  7. 7.

    10× Taq PCR buffer.

     
  8. 8.

    Novex Hi-Density TBE sample buffer (Life Technologies).

     
  9. 9.

    10,000× SYBR Gold nucleic acid gel stain (Life Technologies).

     
  10. 10.

    PCR purification kit.

     
  11. 11.

    Gel extraction kit.

     
  12. 12.

    Restriction enzyme for HR assay depends on the design of the HR template.

     

3 Methods

Ultrapure water is used for all the steps below. The setup of the experiments is carried out at room temperature unless otherwise stated.

3.1 Design and Cloning of Genome Engineering Constructs

Below is a step-wise protocol for cloning and verification of targeting vectors. Prior to the cloning of the constructs for genome engineering, the target sites should be selected as described in Subheading 4 (see Notes 1 3 ).
  1. 1.

    Prepare sgRNA oligo duplex.

    Resuspend the oligos (see Note 2 ) to a final concentration of 100 μM with TE buffer. Mix regents listed below to prepare the reaction mixture for each pair of oligos to phosphorylate and anneal them into ligation-ready duplex DNA fragments.

    1 μl

    forward oligo (100 μM)

    1 μl

    reverse oligo (100 μM)

    1 μl

    10× T4 Ligation Buffer

    6 μl

    ddH2O

    1 μl

    T4 PNK (NEB)

    10 μl

    total volume

    Place the mixture in a thermocycler using the following parameters to phosphorylate and anneal the oligos.

    37 °C

    30 min

    95 °C

    5 min and then ramp down to 25 °C at 5 °C/min

    4 °C

    hold until ready to proceed

    Add 2 μl of annealed oligos into 398 μl of water to dilute the products 200-fold.

     
  2. 2.

    Cloning of targeting constructs.

    Select the appropriate cloning backbone vector to insert the annealed oligos (see Notes 2 and 4 ). Mix reagents listed below to set up the ligation reaction. Mix the same reagents without adding the sgRNA oligo duplex to prepare a negative control reaction.

    X μl

    backbone plasmid (total amount 100 ng)

    2 μl

    sgRNA oligo duplex (200-fold diluted)

    2 μl

    10× Tango buffer

    1 μl

    DTT

    1 μl

    ATP

    1 μl

    FastDigest BbsI

    0.5 μl

    T7 ligase

    Y μl

    ddH2O

    20 μl

    total

    Incubate the ligation reaction in a thermocycler following the parameters below.

    37 °C

    5 min

    23 °C

    5 min

    Cycle the previous two steps for 6 cycles (total run time 1 h)

     

    4 °C

    hold until ready to proceed

     
  3. 3.

    (Optional but highly recommended) Plasmid-Safe treatment.

    Add Plasmid-Safe buffer, ATP, and the ATP-dependent Plasmid-Safe exonuclease as listed below to treat the ligation reaction and the negative control to increase the efficiency of cloning by degrading unligated DNA fragments in the product mixture.

    11 μl

    ligation reaction from previous step

    1.5 μl

    10× Plasmid-Safe Buffer

    1.5 μl

    10 mM ATP

    1 μl

    ATP-dependent Plasmid-Safe exonuclease

    15 μl

    total

    Incubate the reaction mixture in a thermocycler following the parameters below.

    37 °C

    30 min

    70 °C

    30 min

    4 °C

    hold until ready to proceed

     
  4. 4.

    Transformation.

    Add 2 μl of each reaction from previous step (including the negative control ligation reaction) into chosen competent cells, such as Stbl3, transform following the manufacturer’s protocol.

    Plate the transformed cells on Ampicillin selection LB agar plates or other type of plates depending on the selection marker of the backbone vector.

     
  5. 5.

    Plasmid preparation.

    Check the plate for presence of bacterial colonies after overnight incubation. Usually there should be no or less than five colonies on the negative control plate, whereas over tens to hundreds of colonies will grow on the cloning plate, indicating high cloning efficiency.

    Pick two to three colonies from each agar plate and set up a small volume (typically 5 ml) LB culture for miniprep. Incubate and shake under 37 °C overnight, prepare plasmid DNA using a spin miniprep kit according to manufacturer’s protocol.

     
  6. 6.

    (Optional) Digest the plasmid DNA to verify the insertion of the oligos.

    Digest the miniprep DNA with diagnostic restriction enzymes, BbsI and AgeI, if using the vector backbone PX330/PX335. Run the digested product on a 1 % agarose gel to visualize the band pattern of the digested product.

    When a lot of cloning is done at the same time, it is possible to screen for correct insertion of the target sequence oligos by this digestion because a successful insertion will destroy the BbsI sites. After double digestion, clones with insertion of annealed sgRNA oligos will show only linearized plasmid, whereas clones without insertion will yield two fragments, with sizes of ~980 and ~7,520 bp in the case of pX330 when running and visualizing on an agarose gel.

     
  7. 7.

    Sequencing verification of positive clones.

    Sequence the clones with a forward primer that binds the human U6 promoter to verify the successful insertion of guide sequences. The verified clones can be then prepared using a Midi or Maxi plasmid extraction kit for downstream experiments.

     

3.2 Mammalian Cell Culture and Transfection

As a general protocol, the steps below use HEK 293FT cells as an exemplar system to demonstrate CRISPR-Cas9 genome engineering. Additional suggestions can be found in Subheading 4 (see Note 5 ).
  1. 1.

    HEK 293FT cell maintenance.

    Maintain the HEK cells in DMEM medium supplemented with 10 % FBS (D10 medium) in an incubator at 37 °C temperature and supplemented with 5 % CO2 as recommended by the manufacturer. Feed the cells every other day, and passage the cells so they never reach over 75 % confluence.

     
  2. 2.

    Preparing cells for transfection.

    At 16–24 h before transfection, dislodge and disassociate the cells by trypsinization. Plate cells into 24-well plates containing 500 μl antibiotic free medium at a density of 250,000 cells per mL of culture medium, or 125,000 cells per well. Scale up or down the culture volume proportionally to the plate format based on the number of cells needed in the experiment. At the time of transfection, the cells should be at around 75–85 % confluence.

     
  3. 3.

    Transfection.

    Mix a total of up to 600 ng DNA containing the targeting constructs in a microcentrifuge tube for each well of transfection on a 24-well plate. For transfecting plasmid derived from pX330 or pX335, where a guide sequence has been cloned into the backbone vector, directly use 600 ng of the prepared plasmid in the transfection mixture. When transfecting more than one construct for alternative designs, e.g., the double nickase method, or when multiplex genome cleavage is desired, mix equal molar ratio of all constructs to a total of 600 ng in the transfection mixture (see Notes 2 and 4 ). Add 50 μl OptiMEM medium OptiMEM medium to the DNA mixture. Mix well and spin down.

    Dilute Lipofectamine 2000 transfection reagent by adding 1.5 μl of the reagent into 50 μl of OptiMEM, Mix well and incubate at room temperature for 5 min. Within 15 min of diluting the transfection reagent, add all the diluted Lipofectamine 2000 reagent into the DNA-OptiMEM mixture prepared earlier. Mix well and spin down. Incubate the mixture for another 20 min to allow the formation of DNA–Lipofectamine complex. Add the final complex directly onto the culture medium of each well on the 24-well plate. (Not required but highly recommended) Include a transfection control to monitor transfection efficiency. Use a control plasmid expressing a fluorescent protein, such as pCMV-EGFP, and transfect this plasmid following same protocol as above into the cell. Transfect another well of cells using the SpCas9 backbone vector, e.g. pX330 or pX335, as a negative control for downstream processing and validation of assay.

    Replace the medium with warmed fresh medium around 12–24 h post transfection. Maintain the cells for another 48–72 h to allow sufficient time for genome engineering mediated by CRISPR-Cas9 system.

     

3.3 Genome Cleavage Analysis

  1. 1.

    Genomic DNA extraction.

    Extract genomic DNA from transfected cells using the QuickExtract DNA extraction kit following the manufacturer’s recommended protocol. Briefly, disassociate cells from the plate, harvest by spin down the cell suspension at 250 × g for 5 min. Wash cell pellet with 500 μl of PBS and then resuspended in QuickExtract solution. We typically use 50 μl for one well in a 24-well plate (scale up and down accordingly). Vortex the suspension and incubate at 65 °C for 15 min, 68 °C for 15 min, and 98 °C for 10 min.

     
  2. 2.

    SURVEYOR assay to detect genomic cleavage.

    Use SURVEYOR assay to detect genomic cleavage after extraction of genomic DNA (see Note 6 ), following the stepwise instructions provided in the SURVEYOR Mutation Detection Kit manual. A brief description of the steps is listed below.
    1. (a)

      Amplify extracted genomic DNA with a pair of primers designed for the target region of interest using a high-fidelity enzyme such as Herculase II fusion polymerase of Kapa Hifi Hotstart polymerase. Typically an amplicon size of less than 1,000 bp is preferred as shorter amplicon gives more specific amplification products.

       
    2. (b)

      Visualize the PCR product on an agarose gel to check the specificity of the amplification. It is important to have very specific amplification of genomic region to yield accurate SURVEYOR assay results as nonspecific bands will interfere with the interpretation of gel electrophoresis analysis.

       
    3. (c)

      Purify and quantify the PCR products, set up a denaturing/re-annealing reaction by mixing up to 400 ng of PCR products with water and re-annealing buffer (we typically use the PCR reaction buffer and add to a final concentration of 1×, refer to the SURVEYOR assay manual for more information).

       
    4. (d)
      Run the reaction in a thermocycler with following parameters.

      95 °C

      5 min and then ramp down to 85 °C at −2 °C/s

      85 °C

      1 min and then ramp down to 25 °C at −0.1 °C/s

      4 °C

      hold until ready to proceed

       
    5. (e)

      Digest the re-annealed products with SURVEYOR enzyme kit at 42 °C for 1 h as recommended by the manufacturer’s protocol.

       
    6. (f)

      Visualize the digested product using gel electrophoresis.

      For visualization of SURVEYOR assay results, we recommended loading of the Surveyor Nuclease digestion products with Polyacrylamide gel electrophoresis (PAGE) method as it gives better solution compared with agarose gels.

      Quantification of the assay results and the method to convert it to an estimation of the frequency of indels generated by CRISPR-Cas system in the population of cells are described in Subheading 4 (see Note 7 ).

       
     

3.4 Implementation of Homologous Recombination (HR) Using CRISPR-Cas System

The guideline and considerations for designing a HR experiment to use CRISPR-Cas system to precisely modify the genomic sequence of interest by inserting, deleting, or replacing part of the genome are described in Subheading 4 (see Note 8 ). Briefly, following design and cloning of the HR template, perform HR experiment following steps below.
  1. 1.

    HEK 293FT cell maintenance and the preparation of cells for transfection.

    This part is same as the corresponding steps in Subheading 3.2. Briefly, plate cells into 24-well plates and make sure at the time of transfection, the cells should be at around 75–85 % confluence.

     
  2. 2.

    Transfection.

    Mix a total of up to 800 ng DNA containing the targeting constructs and the HR template vector (or single-stranded DNA oligos, see Note 9 ) in a microcentrifuge tube for each well of transfection on a 24-well plate. Generally, apply a molar ratio of 1:3–5:1 for the targeting vector and HR template vector. (Optional but recommended) Titrate different molar ratio between the targeting vector and HR template vector to test the optimal condition for the HR experiment. Add 50 μl OptiMEM medium to the DNA mixture. Mix well and spin down.

    Dilute Lipofectamine 2000 transfection reagent by adding 2 μl of the reagent into 50 μl of OptiMEM, Mix well and incubate at room temperature for 5 min. Within 15 min of diluting the transfection reagent, add all the diluted Lipofectamine 2000 reagent into the DNA-OptiMEM mixture prepared earlier. Mix well and spin down. Incubate the mixture for another 20 min to allow the formation of DNA–Lipofectamine complex. Add the final complex directly onto the culture medium of each well on the 24-well plate.

    Transfect another well of cells using the SpCas9 backbone vector, e.g., pX330 or pX335, together with the same amount of HR template vector as a negative control.

    Replace the medium with warmed fresh medium around 12–24 h post transfection. Maintain the cells for another 72 h to allow sufficient time for HR mediated by CRISPR-Cas9 system and the template.

     

3.5 Verification and Quantification of HR Efficiency

To verify the homologous recombination between the HR template and the endogenous genome, restriction fragment length polymorphism (RFLP) assay for HR can be applied.
  1. 1.

    Genomic DNA extraction.

    Extract the genomic DNA using the same extraction protocol using the QuickExtract DNA extraction kit as in the SURVEYOR assay (Subheading 3.3).

     
  2. 2.

    Target region amplification.

    Amplify the genomic region of interest by a HR testing primer set where the two primers bind outside the homology region to avoid false positive results given by amplification of the residue HR template.

     
  3. 3.

    Perform RFLP digestion.

    Run the resulting PCR product on an agarose gel to check for specificity of amplification, as nonspecific PCR products will interfere with the assay and prevent accurate quantification of HR efficiency. In many cases, several pairs of HR testing primer sets should be screened to obtain robust, specific amplicons.

    Purify the PCR amplification product by standard PCR purification, or in the case where clean PCR product cannot be obtained, gel extract the desired amplicon following separation of PCR product on an agarose gel.

    Digest the purified products with the appropriate enzyme corresponding to the design of the HR template (see Note 8 ), and visualize on an agarose gel or PAGE gel. The latter usually gives better resolution and is highly recommended. The efficiency of HR in the population of cells assayed can be estimated by the following formula:

    HR percentage (%) = (m + n/m + n + p) × 100

    Here the number “m” and “n” indicate the relative quantity of bands from digested genomic PCR products, whereas the “p” equals the relative quantity of undigested products.

     
  4. 4.

    Perform additional Sanger and next-generation DNA sequencing to verify the presence of desired engineered sequences within the genome. Briefly, clone the genomic PCR product into a sequencing vector, TOPO-TA, or other blunt-end cloning method, and perform Sanger sequencing to detect recombined genomic amplicons. Alternatively for higher throughput, subject the genomic PCR products to next-generation sequencing.

     

4 Notes

  1. 1.

    Identification and selection of target genomic site. The two primary rules for identifying a target site for the SpCas9 system are: (1) finding the “NGG” PAM sequence which is required for SpCas9 targeting, and (2) picking a sequence of 20 bp in length upstream of the PAM to its 5′ end as the guide sequence. Following these two guidelines, multiple potential target sites can be usually found within the genomic region of interest (Fig. 2). Additionally, when using U6 promoter to express sgRNA (such as pX330, pX335), we suggest adding the G (not replacing but add one more base) because the human U6 promoter requires a “G” at the transcription start site to have highest level of expression (Fig. 2). While we do notice that sometimes the sgRNA will still work without the extra “G”, it is generally better to have this additional base. In the case where the guide sequence starts with a base “G”, this addition can be omitted. In our open-source online resources website (http://crispr.genome-engineering.org), we provide the most up-to-date information for using the CRISPR-Cas9 system for genome engineering, focusing on the SpCas9 system. Additionally, we also developed an online tool for the selection of SpCas9 targets for different organisms including human, mouse, zebrafish, C. elegans, etc. This tool can greatly facilitate and simplify the process of performing target selection in batch (http://crispr.mit.edu/). Because the efficiency of different targets could vary considerably depending on the guide sequences, we highly recommend testing multiple target sites for each gene or region of interest and selecting the most effective target (see Note 1 ). In the case of double nickase design, we recommend individually testing each target with the wild type SpCas9 system to assess the cleavage efficiency of individual guides and then combine the most efficient pair of guides with opposite directionality and appropriate spacing for the genome engineering application.

     
  2. 2.
    Design of oligos for inserting guide sequences into backbone vectors. The cloning vectors we use for typical SpCas9 genome targeting are pX330 for the wild type SpCas9 and pX335 for the nickase version SpCas9n. Both vectors are mammalian dual-expression vectors, which enables the co-expression of SpCas9 protein driven by the potent constitutive promoter CBh and sgRNA driven by the RNA Pol III human U6 promoter in mammalian cells (Fig. 3). CBh promoter is a hybrid promoter derived from the CAG promoter, which have been validated to support strong expression of transgene in multiple cell types/lines, including HEK 293FT, mouse Neuro-2a, mouse Hepa1-6, HepG2, HeLa, human ESCs, and mouse ESCs. To clone custom guide sequences into these backbone vectors, a pair of oligos encoding the guide sequences can be ordered with the appropriate overhangs (Fig. 3), then annealed to form a clone-ready duplex DNA fragment. The vector can be then digested using BbsI, and a pair of annealed oligos can be cloned into the backbone to express the corresponding sgRNA (Fig. 3). The oligos are designed based on the target site sequence selected in previous section. A common confusion sometimes in cloning the guide into backbone vector is to include the “NGG” PAM sequence in the guide sequence. Hence, it is important to check that only the 20 bp sequence to the 5′ end of the PAM is being used for designing the oligo sequences to order. An alternative way of designing oligos for directly amplifying a PCR fragment that contains the U6 promoter driving a sgRNA could also be employed for testing the guide sequences, which simplifies the test by avoiding the need of cloning, but might be less efficient than using the cloned vector plasmids (see Note 4 , [41]).
    Fig. 3

    Schematics for the cloning backbone vectors pX330/pX335 with oligo design for inserting guide sequences. The pX330 and pX335 vectors contains dual-expression cassettes for both the SaCas9 protein and the sgRNA. Digestion of the backbone with BbsI Type II restriction sites (blue) generates the complementary cloning overhangs to the annealed oligos (purple boxed). Note that a G–C base pair is added at the 5′ end of the guide sequence for optimal U6 transcription. The oligos contain overhangs for ligation into the overhangs of BbsI sites. The top and bottom strand orientations is exactly identical to those of the genomic target but exclude the “NGG” PAM. Parts of this figure are adapted from [19, 41]

     
  3. 3.

    Screening of multiple guides. For most applications, we screen for at least three guide sequences within the target genomic region in an effort to find the most efficient ones. This is because while CRISPR-Cas9 system works very efficiently, the actual cleavage efficiency could be affected by the sequence of the guide, the accessibility of local chromatin, the activity of the endogenous DNA repair pathways, and other guide-specific or cell-type-specific factors. Hence, to ensure that a valid guide sequence is obtained, this screening process is highly recommended. Following the same logic, a guide sequence that has been verified in one cell type will not necessarily work to the same efficiency in another cell type or condition. Hence, additional optimization or re-screening of new guide sequences might be required when moving from one experimental system to another. This same situation is also applicable to the HR experiment where the HDR efficiency can very considerably among different types of cells or tissues.

     
  4. 4.

    Additional strategy for screening guides and backbones for different applications. We have also developed another way of quickly screening guide sequences with amplified PCR products. In this design, two primers are used to amplify the U6 RNA-expression promoter, where the forward primer binds to the 5′ beginning of U6 promoter, and the reverse primer binds to the 3′ end of the U6 promoter. Because the reverse primer also contains a long extension that can add on the guide sequence and the chimeric sgRNA scaffold, the amplified PCR product contains all necessary elements for expressing a sgRNA containing the guide specified in the reverse primer. Hence, the screening of guide sequences can be done by co-transfecting this PCR product with a backbone vector expressing the SaCas9 protein. Because many application of CRISPR-Cas9 genome engineering involve cell lines that might be difficult to work with, e.g., cell lines that are hard to transfect, we developed additional backbone vectors to facilitate selection and screening for transfected cells. These vectors contain the fluorescent maker protein, GFP, or the selectable puromycin resistance gene, linked to the expressing of SaCas9 via a 2A peptide linker. These constructs will enable fluorescence activated cell sorting (FACS) or the selection of transfected population, which can further improve the overall efficiency of genome engineering particularly in the case of HR applications. Additional details on these designs and backbones can be found in our recent publication [41].

     
  5. 5.

    Cell line choice for validation of guide design. Functional validation of targeting constructs bearing the designed guides can be carried out in relevant cell lines, e.g., HEK 293FT, K562, Hela for human genome engineering, or Neuro-2a, Hepa1-6 for mouse. This process takes advantage of some favorable experimental properties of these lines, such as robust and easy maintenance, efficient transfection, etc., before embarking on complicated procedures in other mammalian systems. Nonetheless, achieving best results for each experiment might require additional optimization (see Note 2 ). Moreover, due to the genetic and epigenetic differences between cell types or subjects of study, results obtained from one cell type might not necessarily correspond to those from another cell type of the same species (see Note 2 ).

     
  6. 6.

    Mechanism of SURVEYOR nuclease assay. Following the delivery of SpCas9 and the sgRNAs into mammalian cells, the induced genomic cleavage could be assayed by the SURVEYOR assay, which could detect modification of genomic DNA within a population of cells. This assay works when a certain portion of the cells will be modified by SpCas9 so that their genomic sequence at target site is different from the un-modified population. Hence, in the assay, it is possible to amplify region of interest from genomic DNA via PCR, then through a denaturing and re-annealing process to form mismatched DNA. This mismatched DNA can then be recognized by the SURVEYOR nuclease and cleaved for visualization on analytical gels. To quantify the efficiency of genomic cleavage, one can then assess the percentage of cleaved products as a surrogate for the percentage of indels generated within the target genomic region.

     
  7. 7.

    Analysis of SURVEYOR assays results. To calculate the genome cleavage efficiency of a tested target, quantify the band intensity of SURVEYOR assay products visualized by PAGE using the following formula:

    Indel percentage (%) = (1 − √(1 − x)) × 100, where x = (a + b)/(a + b + c)

    In this formula, the number “a” and “b” represent the relative quantities of the cleaved bands, while “c” equals to the relative quantity of the non-cut full-length PCR product.

    Other methodology of detecting the genomic cleavage can also be applied. One such method is to clone the SURVEYOR PCR products into a sequencing vector, e.g., pUC19, and transformed into E. coli. These individual clones can be then sequenced via Sanger sequencing to reveal the identity of genome modifications. Additionally, the percentage of modified clones can also be used as a measurement for the efficiency of genome engineering. Alternatively, the PCR products could also be sequenced in a more high-throughput way with next-generation sequencing.

     
  8. 8.
    Design and synthesis of repair template for HR experiment. For introducing a precise genomic modification into the genome, the HDR pathway can be employed. This is achieved by co-transfecting SpCas9 constructs (derived from pX330 or pX335) bearing guide sequences with a HR template in the target cell line. After recombination, modifications such as point mutation, small and large insertions/deletions, or other type of chromosomal changes could be engineered into the endogenous genome. A few considerations for the choice of guide:
    1. (a)

      Typically, a screening for the most efficient guide sequence is performed first. We recommend picking several (three to six) targets within the genomic region of interest following protocols listed earlier. Tests are then performed to assay the cleavage efficiency of each of these guides. Then, the actual HR experiments can be carried out with the most efficient guides (also see additional considerations in Notes 3 and 4 ).

       
    2. (b)

      For maximize the efficiency of HR, it is recommended that the cleavage site of the guide is as close to the junction of the homology arm, i.e., the size at which genome modifications are introduced, as possible. Usually this distance should be less than 100 bp, ideally less than 10 bp.

       
    3. (c)

      To minimize the off-target cleavage, the double nickase design can be used. In this case, multiple guide sequences can be first tested individually, and typically the combination of highest cutting guide designs with appropriate directionality will yield highest cleavage when used in the paired fashion, thus giving best results in HR experiment.

       
     
  9. 9.
    The HR template is essentially the desired sequence that needs to be present in the engineered genome, flanked by two homology arms bearing the same sequence as the reference genome. Below are considerations for the choice of HR template:
    1. (a)

      It is usually advised to insert a testable marker in the HR template to facilitate the assay for successful HR events. For example, a restriction site could be inserted to allow RFLP assay. Alternatively, the insertion of fluorescent proteins or selectable drug-resistance genes such as puromycin-resistance cassette can also be used.

       
    2. (b)

      For introducing single-point mutation the best HR template for transfection is usually single-stranded DNA (ssDNA) oligos. For ssDNA oligo design, we typically use around 50–90 bp homology arms on each side and introduce your mutation/modification in between the two arms. When ordering long oligos, ultramer oligo (IDT) is recommended.

       
    3. (c)

      For introducing larger genomic modification, plasmid DNA vector can be used because of the length limit of ssDNA oligos. When designing a plasmid-based HR template, a minimum of 800 bp homology arms on each side is recommended.

       
    4. (d)

      If you have intact “protospacer + PAM” sequence within the HR template, it can lead to the HR template being degraded by Cas9. Hence, it is recommended to make silent mutations to destroy the sgRNA-binding site, or avoid putting in the full target site in the HR template by choosing target sites that span the site of modification. For making silent mutations, one good option is to mutate the PAM “NGG” within the HR template, as the PAM is required for cleavage. For example, change the “NGG” to “NGT” or “NGC”, in addition to mutations in the spacer itself, could usually prevent degradation of donor plasmid.

       
     

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Broad Institute of MIT and HarvardCambridgeUSA
  2. 2.McGovern Institute for Brain Research, Department of Brain and Cognitive SciencesDepartment of Biological Engineering, Massachusetts Institute of TechnologyCambridgeUSA

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