1. Design of sgRNAs
Cas Endonuclease | PAM | Optimal spacer length |
SpCas9 | NGG | 17-22 nt inclusive |
SaCas9 | NNGRRT | ≥ 21 nt |
NmCas9 | NNNNGATT | ≥ 19 nt |
AsCas12a and LbCas12a | TTTV | ≥ 19 nt |
Table 1: Some commonly used Cas enzymes with their cognate PAMs and optimal sgRNA lengths. N = Any nucleotide (A, T, G, or C); R = A or G; V = A, C, or G.
CRISPR Plasmid | Sequence |
pSpCas9 and pSaCas9 | Sense: 5' – CACC(G)NNNNNNNNNNNNNNNNNNNNN – 3' Antisense: 3' – (C)NNNNNNNNNNNNNNNNNNNNNCAAA – 5' |
pNmCas9 | Sense: 5' – CACC(G)NNNNNNNNNNNNNNNNNNNNN – 3' Antisense: 3' – (C)NNNNNNNNNNNNNNNNNNNNNCAAC – 5' |
pAsCas12a and pLbCas12a | Sense: 5' – AGATNNNNNNNNNNNNNNNNNNNNN – 3' Antisense: 3' – NNNNNNNNNNNNNNNNNNNNNAAAA – 5' |
Table 2: Oligonucleotides required for cloning sgRNA sequences into CRISPR plasmids used in a recent evaluation study25. The overhangs are italicized.
Figure 2: An example illustrating how to select target sites and design oligonucleotides for cloning into CRISPR plasmids. The target genomic locus here is exon 45 of the human CACNA1D gene. The PAMs for SpCas9 and SaCas9 are NGG and NNGRRT respectively and are highlighted in red, while the PAM for AsCas12a and LbCas12a is TTTN and is highlighted in green. The red horizontal bar indicates the protospacer for SpCas9 and SaCas9, while the green horizontal bar indicates the protospacer for the two Cas12a enzymes. Please click here to view a larger version of this figure.
2. Cloning of oligonucleotides into a backbone vector
Figure 3: An example of a CRISPR plasmid. (a) A map indicating different important features of the plasmid. Here, the EF-1a promoter drives the expression of Cas9, while the U6 promoter drives the expression of the sgRNA. Amp(R) indicates an ampicillin-resistance gene in the plasmid. (b) The sequence of the “BbsI-BplI cloning site” in the plasmid. The recognition sequence of BbsI is GAAGAC and is indicated in red, while the recognition sequence of BplI is GAG-N5-CTC and is indicated in green. (c) Primers that can be used for colony PCR to check whether the sgRNA sequence has been successfully cloned into the plasmid. The hU6_forward primer is indicated by a purple arrow on the plasmid map, while the universal M13R(-20) primer is indicated by a pink arrow on the plasmid map. Please click here to view a larger version of this figure.
3. Design and synthesis of repair templates
NOTE: For precision genome engineering, a template specifying the desired DNA modifications needs to be provided together with the CRISPR reagents. For small DNA edits such as alteration of a single nucleotide, ssODN donor templates are most suitable (see section 3.1). For larger DNA edits such as insertion of a GFP tag 5’ or 3’ of a particular protein-coding gene, plasmid donor templates are most suitable (see section 3.2).
Figure 4: Design of ssODN donor templates. (a) Schematic illustrating various possible designs. The red horizontal rectangles indicate the non-target (NT) strand, while the blue rectangles indicate the target (T) strand. In addition, the small green rectangles indicate the desired DNA modifications (such as single nucleotide changes). When a symmetric ssODN is used, the minimum length of each homology arm should be at least 17 nt (but can be longer). For asymmetric ssODNs, the 37/77 T ssODN appears to be optimal for SpCas9-induced HDR, while the 77/37 NT ssODN appears to be optimal for Cas12a-induced HDR. L = left homology arm; R = right homology arm. (b) A specific example to demonstrate how to design ssODN templates. Here, the target genomic locus is exon 45 of the human CACNA1D gene. The PAM for Cas9 is pink and underlined, while the PAM for Cas12a is brown and underlined. The goal is to create a missense mutation (highlighted in green) by converting AGU (encoding serine) to AGG (encoding arginine). To prevent re-targeting by Cas12a, the TTTC PAM is mutated to CTTC. Note that there is no change in amino acid (UAU and UAC both code for tyrosine). To further prevent re-targeting by Cas9, an AGU codon is replaced with a UCC codon (bold), both of which code for serine. Please click here to view a larger version of this figure.
Figure 5: Design and cloning of a plasmid donor template. (a) The goal in this specific example is to fuse P2A-GFP to the C-terminus of the CLTA protein. The blue horizontal rectangle indicates the left homology arm, while the red horizontal rectangle indicates the right homology arm. Capital letters indicate protein-coding sequences, while lowercase letters indicate non-coding sequences. The PAMs for SpCas9 and Cas12a are italicized and underlined. (b) A plasmid donor template that can be used to endogenously tag P2A-GFP at the C-terminus of CLTA. The provided primer sequences can be used to clone the plasmid by Gibson assembly. The PCR conditions are as follows: 98 °C for 3 min, 98 °C for 30 s (step 2), 63 °C for 30 s (step 3), 72 °C for 1 min (step 4), repeat steps 2‒4 for another 34 cycles, 72 °C for 3 min, and hold at 4 °C. Black letters correspond to vector sequences, blue letters correspond to the left homology arm, green letters correspond to P2A-GFP, and red letters correspond to the right homology arm. Note that once the sequence encoding P2A-GFP is successfully integrated into the target locus, re-targeting by SpCas9 will not be possible, since only 9 nt of its protospacer (GTGCACCAG) will be left intact. Moreover, in order to prevent re-targeting by Cas12a, three basepairs immediately downstream of the STOP codon (in bold) are deleted from the plasmid sequence. Please click here to view a larger version of this figure.
4. Cell transfection
NOTE: The remaining parts of the protocol are written with HEK293T cells in mind. The culture medium used consists of Dulbecco Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose, 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 0.1% penicillin/streptomycin. Different steps of the protocol may have to be modified according to the actual cell line used. All cell culture work is done in a Class II Biosafety Cabinet to ensure a sterile work environment.
5. Fluorescence activated cell sorting (FACS) of transfected cells
6. Expansion of individual clones
7. Evaluation of editing efficiency
Figure 6: Checking cells for successful genome editing outcomes. (a) A schematic illustrating two commonly used assays, namely the mismatch cleavage assay with the T7 endonuclease I (T7EI) enzyme and next generation sequencing (NGS) or targeted amplicon sequencing. The blue horizontal rectangles indicate DNA and the yellow circles indicate modifications induced by the CRISPR-Cas system. Primers for the T7E1 assay are denoted in green, while primers for generating amplicons for NGS are denoted in red. (b) Design of primer sequences for the T7EI cleavage assay and for NGS. Here, the target genomic locus is exon 45 of the human CACNA1D gene. The intended modification site is indicated by an asterisk. Please click here to view a larger version of this figure.
8. Screening of individual clones
To perform a genome editing experiment, a CRISPR plasmid expressing a sgRNA targeting the locus-of-interest needs to be cloned. First, the plasmid is digested with a restriction enzyme (typically a type IIs enzyme) to linearize it. It is recommended to resolve the digested product on a 1% agarose gel alongside an undigested plasmid to distinguish between a complete and partial digestion. As undigested plasmids are supercoiled, they tend to run faster than their linearized counterparts (see Figure 7a). Second, two oligonucleotides for the sgRNA have to be annealed and ligated into the cut plasmid. To determine if the oligonucleotides have been successfully cloned into the CRISPR plasmid backbone, colony PCR is performed (see Figure 7b). Positive clones are then inoculated before the plasmids are extracted and sent for Sanger sequencing.
Figure 7: Cloning of sgRNA-expressing CRISPR plasmids. (a) Image showing digested and undigested plasmids after gel electrophoresis. Lanes 1‒3 show plasmids that have been linearized completely by BbsI. Lane 4 shows the original undigested plasmid, which is supercoiled and hence migrates faster on an agarose gel. (b) Representative gel image of colony PCR products. Positive clones are marked by a green tick (indicating that oligonucleotides have been successfully ligated into the vector backbone), while negative clones are marked by a red cross. Please click here to view a larger version of this figure.
Once the constructs have been sequence-verified, they can be transduced into a desired cell line (such as HEK293T). CRISPR plasmids often carry a selectable marker (e.g., an mCherry gene), thereby allowing successfully transduced cells to be selected. Fluorescence can be readily visualized under a microscope upon successful delivery of the plasmids (see Figure 8a). The cells are sorted by flow cytometry around 24‒72 h post-transfection. The gating is set based on a non-transfected control (see Figure 8b). Part of the sorted cells are seeded on a tissue culture plate to allow for recovery.
Figure 8: Introduction of CRISPR reagents into human cells. (a) Representative microscopy image of HEK293T cells successfully transfected with a CRISPR plasmid bearing a mCherry marker (see the Table of Materials). Transfection efficiency can be estimated by the percentage of cells displaying red fluorescence. 10x magnification, scale bar represents 200 µm. (b) Representative FACS plots. Transfected cells (right panel) are gated against a non-transfected control (left panel). Please click here to view a larger version of this figure.
While the sorted cells are recovering, the editing efficiency is evaluated to determine whether the experiment should be continued. Genomic DNA (gDNA) is isolated from the remaining un-plated cells. A T7EI assay is performed to check the cleavage efficiency, which is calculated from the intensities of the bands observed on an agarose gel (see Figure 9a). Additionally, if an HDR-based experiment is designed to incorporate a restriction site at the target locus, a RFLP assay can be performed with the corresponding restriction enzyme (see Figure 9b).
Figure 9: Evaluating the extent of genome editing. (a) Representative gel image showing the results of a T7E1 assay. Lanes 1‒8 represent different experimental samples. For each sample, the top band indicates uncut DNA, while the bottom two bands indicate the cleavage products. Varying NHEJ efficiencies can be observed among the samples. Digestion of PCR amplicons from non-transfected cells is used as a negative control and is marked by a "-". (b) Representative gel image showing the results of a RFLP assay. Lanes 1‒6 represent different experimental samples. For each sample, the top band indicates uncut DNA, while the bottom two bands indicate the cleavage products after restriction digest. Varying HDR efficiencies can be observed among the samples. Please click here to view a larger version of this figure.
If the editing efficiency is acceptable (e.g., at least 5% by the T7EI assay), we proceed to identify individual clones that carry the desired DNA modifications. Plated cells, which have been left to recover, are seeded sparsely to allow for individual colonies to grow. Subsequently, individual colonies are picked and expanded, before gDNA is isolated from these individual clones. Another round of T7EI assay is performed to identify clones with modified DNA at the target site. TOPO cloning and Sanger sequencing are then performed to determine the exact sequences of all alleles. Ideally, for gene knockouts, the indels should not be in multiples of three, which do not cause frameshift mutations (see Figure 10a). To further validate that a protein-coding gene has been successfully inactivated, a western blot can be carried out to ensure that no targeted protein is present (see Figure 10b).
Figure 10: Screening for gene knockouts. (a) Representative Sanger sequencing data. The original unmodified DNA sequence (top row) can be aligned with the sequencing result received (bottom row) to check whether any edits have taken place. Here, there is a 1 bp insertion and a 7 bp deletion at the target site (as depicted by the red boxes, which represent gaps in the alignment). (b) Representative western blot image. Here, several clones (expanded from single cells) are screened for the presence or absence of the GLUL protein. The red arrows indicate the clones where the GLUL gene has been successfully knocked out. “WT” stands for wildtype cells, where GLUL is highly expressed. ACTB (β-actin) serves as a loading control (see the Table of Materials). Please click here to view a larger version of this figure.
T4 Polynucleotide Kinase (PNK) | NEB | M0201 | |
Shrimp Alkaline Phosphatase (rSAP) | NEB | M0371 | |
Tris-Acetate-EDTA (TAE) Buffer, 50X | 1st Base | BUF-3000-50X4L | Dilute to 1X before use. The 1X solution contains 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA. |
Tris-EDTA (TE) Buffer, 10X | 1st Base | BUF-3020-10X4L | Dilute to 1X before use. The 1X solution contains 10 mM Tris (pH 8.0) and 1 mM EDTA. |
BbsI | NEB | R0539 | |
BsmBI | NEB | R0580 | |
T4 DNA Ligase | NEB | M0202 | 400,000 units/ml |
Quick Ligation Kit | NEB | M2200 | An alternative to T4 DNA Ligase. |
Rapid DNA Ligation Kit | Thermo Scientific | K1423 | An alternative to T4 DNA Ligase. |
Zero Blunt TOPO PCR Cloning Kit | Thermo Scientific | 451245 | The salt solution comes with the TOPO vector in the kit. |
NEBuilder HiFi DNA Assembly Master Mix | NEB | E2621L | Kit for Gibson assembly. |
One Shot Stbl3 Chemically Competent E.Coli | Thermo Scientific | C737303 | |
LB Broth (Lennox), powder | Sigma Aldrich | L3022 | Reconstitute in ddH20, and autoclave before use |
LB Broth with Agar (Lennox), powder | Sigma Aldrich | L2897 | Reconstitute in ddH20, and autoclave before use |
SOC media | – | – | 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose in 1 L of LB Broth |
Ampicillin (Sodium), USP Grade | Gold Biotechnology | A-301 | |
REDiant 2X PCR Mastermix | 1st Base | BIO-5185 | |
Agarose | 1st Base | BIO-1000 | |
T7 Endonuclease I | NEB | M0302 | |
Plasmid DNA Extraction Miniprep Kit | Favorgen | FAPDE 300 | |
Dulbecco's Modified Eagle Medium (DMEM), High Glucose | Hyclone | SH30081.01 | 4.5 g/L Glucose, no L-glutamine, HEPES and Sodium Pyruvate |
L-Glutamine, 200mM | Gibco | 25030 | |
Penicillin-Streptomycin, 10, 000U/mL | Gibco | 15140 | |
0.25% Trypsin-EDTA, 1X | Gibco | 25200 | |
Fetal Bovine Serum | Hyclone | SV30160 | FBS is heat inactivated before use at 56 oC for 30 min |
Phosphate Buffered Saline, 1X | Gibco | 20012 | |
jetPRIME transfection reagent | Polyplus Transfection | 114-75 | |
QuickExtract DNA Extraction Solution, 1.0 | Epicentre | LUCG-QE09050 | |
ISOLATE II Genomic DNA Kit | Bioline | BIO-52067 | An alternative to QuickExtract |
Q5 High-Fidelity DNA Polymerase | NEB | M0491 | |
Deoxynucleotide (dNTP) Solution Mix | NEB | N0447 | |
6X DNA Loading Dye | Thermo Scientific | R0611 | 10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA |
Protease Inhibitor Cocktail, Set3 | Merck | 539134 | |
Nitrocellulose membrane, 0.2µm | Bio-Rad | 1620112 | |
Tris-glycine-SDS buffer, 10X | Bio-Rad | 1610772 | Dilute to 1X before use. The 1x solution contains 25 mM Tris, 192 mM glycine, and 0.1% SDS. |
Tris-glycine buffer, 10X | 1st base | BUF-2020 | Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine. |
Ponceau S solution | Sigma Aldrich | P7170 | |
TBS, 20X | 1st base | BUF-3030 | Dilute to 1X before use. The 1x solution contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. |
Tween 20 | Sigma Aldrich | P9416 | |
Skim Milk for immunoassay | Nacalai Tesque | 31149-75 | |
WesternBright Sirius-femtogram HRP | Advansta | K12043 | |
Antibody for β-actin (C4) | Santa Cruz Biotechnology | sc-47778 | Lot number: C0916 |
MiSeq system | Illumina | SY-410-1003 | |
NanoDrop spectrophotometer | Thermo Scientific | ND-2000 | |
Qubit fluorometer | Thermo Scientific | Q33226 | |
EVOS FL Cell Imaging System | Thermo Scientific | AMF4300 | |
CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458) | Addgene | 48138 | Single vector system: The gRNA is expressed from the same plasmid. |
CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA | Addgene | 61591 | Single vector system: The gRNA is expressed from the same plasmid. |
CRISPR plasmid: xCas9 3.7 | Addgene | 108379 | Dual vector system: The gRNA is expressed from a different plasmid. |
CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9 | Addgene | 42230 | Single vector system: The gRNA is expressed from the same plasmid. |
CRISPR plasmid: hCas9 | Addgene | 41815 | Dual vector system: The gRNA is expressed from a different plasmid. |
CRISPR plasmid: eSpCas9(1.1) | Addgene | 71814 | Single vector system: The gRNA is expressed from the same plasmid. |
CRISPR plasmid: VP12 (SpCas9-HF1) | Addgene | 72247 | Dual vector system: The gRNA is expressed from a different plasmid. |
The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.
The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.
The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.