CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.
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 ability to engineer the genome of any living organism has many biomedical and biotechnological applications, such as the correction of disease-causing mutations, construction of accurate cellular models for disease studies, or generation of agricultural crops with desirable traits. Since the turn of the century, various technologies have been developed for genome engineering in mammalian cells, including meganucleases1,2,3, zinc finger nucleases4,5, or transcription activator-like effector nucleases (TALENs)6,7,8,9. However, these earlier technologies are either difficult to program or tedious to assemble, thereby hampering their widespread adoption in research and the industry.
In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated (Cas) system has emerged as a powerful new genome engineering technology10,11. Originally an adaptive immune system in bacteria, it has been successfully deployed for genome modification in plants and animals, including humans. A primary reason why CRISPR-Cas has gained so much popularity in such a short time is that the element that brings the key Cas endonuclease, such as Cas9 or Cas12a (also known as Cpf1), to the correct location in the genome is simply a short piece of chimeric single guide RNA (sgRNA), which is straightforward to design and cheap to synthesize. After being recruited to the target site, the Cas enzyme functions as a pair of molecular scissors and cleaves the bound DNA with its RuvC, HNH, or Nuc domains12,13,14. The resulting double stranded break (DSB) is subsequently repaired by the cells via either the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. In the absence of a repair template, the DSB is repaired by the error-prone NHEJ pathway, which can give rise to pseudo-random insertion or deletion of nucleotides (indels) at the cut site, potentially causing frameshift mutations in protein-coding genes. However, in the presence of a donor template that contains the desired DNA changes, the DSB is repaired by the high fidelity HDR pathway. Common types of donor templates include single-stranded oligonucleotides (ssODNs) and plasmids. The former is typically used if the intended DNA changes are small (for example, alteration of a single base pair), while the latter is typically used if one wishes to insert a relatively long sequence (for example, the coding sequence of a green fluorescent protein or GFP) into the target locus.
The endonuclease activity of the Cas protein requires the presence of a protospacer adjacent motif (PAM) at the target site15. The PAM of Cas9 is at the 3’ end of the protospacer, while the PAM of Cas12a (also called Cpf1) is at the 5’ end instead16. The Cas-guide RNA complex is unable to introduce a DSB if the PAM is absent17. Hence, the PAM places a constraint on the genomic locations where a particular Cas nuclease is able to cleave. Fortunately, Cas nucleases from different bacterial species typically exhibit different PAM requirements. Hence, by integrating various CRISPR-Cas systems into our engineering toolbox, we can expand the range of sites that may be targeted in a genome. Moreover, a natural Cas enzyme can be engineered or evolved to recognize alternative PAM sequences, further widening the scope of genomic targets accessible to manipulation18,19,20.
Although multiple CRISPR-Cas systems are available for genome engineering purposes, most users of the technology have relied mainly on the Cas9 nuclease from Streptococcus pyogenes (SpCas9) for multiple reasons. First, it requires a relatively simply NGG PAM, unlike many other Cas proteins that can only cleave in the presence of more complex PAMs. Second, it is the first Cas endonuclease to be successfully deployed in human cells21,22,23,24. Third, SpCas9 is by far the best characterized enzyme to date. If a researcher wishes to use another Cas nuclease, he or she would often be unclear about how best to design the experiment and how well other enzymes will perform in different biological contexts compared to SpCas9.
To provide clarity to the relative performance of different CRISPR-Cas systems, we have recently performed a systematic comparison of five Cas endonucleases – SpCas9, the Cas9 enzyme from Staphylococcus aureus (SaCas9), the Cas9 enzyme from Neisseria meningitidis (NmCas9), the Cas12a enzyme from Acidaminococcus sp. BV3L6 (AsCas12a), and the Cas12a enzyme from Lachnospiraceae bacterium ND2006 (LbCas12a)25. For a fair comparison, we evaluated the various Cas nucleases using the same set of target sites and other experimental conditions. The study also delineated design parameters for each CRISPR-Cas system, which would serve as a useful reference for users of the technology. Here, to better enable researchers to make use of the CRISPR-Cas system, we provide a step-by-step protocol for optimal genome engineering with different Cas9 and Cas12a enzymes (see Figure 1). The protocol not only includes experimental details but also important design considerations to maximize the likelihood of a successful genome engineering outcome in mammalian cells.
Figure 1: An overview of the workflow to generate genome edited human cell lines. Please click here to view a larger version of this figure.
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.
The CRISPR-Cas system is a powerful, revolutionary technology to engineer the genomes and transcriptomes of plants and animals. Numerous bacterial species have been found to contain CRISPR-Cas systems, which may potentially be adapted for genome and transcriptome engineering purposes44. Although the Cas9 endonuclease from Streptococcus pyogenes (SpCas9) was the first enzyme to be deployed successfully in human cells21,22,23,24, enzymes from other bacterial species, including those shown in Table 1, have also been shown to function well as engineering tools in human16,26,27,28. Consequently, researchers keen to use the technology are often unsure which system to choose for their work and how to design their experiments for optimal results.
Here, we seek to assist CRISPR users in maximizing their usage of the technology. Fundamental to any genome editing experiment is the selection and cloning of an appropriate sgRNA for targeting. Work done by us and others reveal that different CRISPR-Cas systems are optimally active at different spacer lengths (see Table 1)25. Additionally, the researcher should choose which system to use based on knowledge of the target site. Generally, among the enzymes tested in our evaluation study, SpCas9 and LbCas12a exhibit the highest cleavage efficiency, especially in more highly expressed genes presumably as a result of more open and accessible chromatin. Hence, we recommend these two enzymes for routine genome editing experiments. However, we have found that both nucleases exhibit higher tolerance for mismatches between the sgRNA and targeted DNA than AsCas12a. Hence, if the researcher is targeting a gene with several closely related homologs or a repetitive region of the genome, we recommend using AsCas12a to minimize off-target effects. Alternatively, SaCas9 may also be utilized as it requires longer spacer lengths for robust activity, thereby offering higher intrinsic specificity than SpCas9 and LbCas12a.
Precise editing of a genomic locus requires the introduction of a repair template together with the CRISPR reagents. The template serves as an instruction manual to tell the cells how to repair the double-stranded break via the HDR pathway. Two common forms of template are a ssODN and a plasmid. The design of this repair template is a critical step for optimal HDR efficiency25,39,42,45,46,47. For ssODNs, the DNA strand from which the sequence of the ssODN is derived is important. AsCas12a and LbCas12a exhibit a preference for ssODNs of the non-target strand sequence, while SpCas9 exhibits a preference for ssODNs of the target strand sequence instead. Moreover, HDR efficiencies may potentially be improved further by utilizing asymmetric donors. However, the researcher has to be careful of which homology arm is extended (see Figure 2). Increasing the length of the wrong arm will end up degrading the HDR efficiency instead. For plasmid donors, the template is often linearized by restriction digest before cell transduction. In addition, a “double-cut” plasmid donor, where spacer-PAM sequences are placed before and after the left and right homology arms respectively, has been shown to yield a higher HDR efficiency than a plasmid donor without the additional spacer-PAM sequences42.
Besides preparation of the necessary CRISPR reagents with or without a repair template, generation of modified cell lines (e.g., containing specific gene knockouts) also involves cell transduction, single cell expansion, and screening of individual clones. The protocol outlines all the downstream steps to enable users to perform a complete genome editing experiment. Some details are cell line-specific. For example, some cell lines may be easy to transfect, while others may require extensive optimization with different transduction methods, including nucleofection and electroporation. Importantly, from our experiences, a critical step in the protocol is single-cell cloning. Certain cell types may not proliferate when they are seeded as a single cell in a well, perhaps due to a lack of secreted growth factors. For example, human embryonic stem cells and many patient-derived primary cell lines typically grow very poorly as isolated single cells. Hence, after transduction, we recommend plating cells sparsely and then picking individual colonies on a plate over using flow cytometry to sort single cells into different wells of a 96-well plate. There may also be a need to add pro-survival factors to the cell culture media, such as the ROCK inhibitor48, to improve the recovery of dissociated cells. In addition, during the process of single-cell cloning, there may be occasions where two separate cells grow very close to each other and end up merging, thereby giving the false appearance of one colony only. In such situations, during the stages of determining the genomic mutations introduced, one may end up identifying three or more distinct alleles. Nevertheless, it is still possible to recover individual clones by sparsely plating these cells again and then picking additional individual colonies.
In conclusion, the CRISPR-Cas technology is revolutionizing biomedical and biotechnological research. However, it currently suffers from several limitations (such as restricted targeting range and large protein size) that hampers its widespread adoption in some applications, including gene therapy. Nevertheless, as time progresses, more and more naturally occurring Cas enzymes will be discovered in a wide range of microbial species and some of these new enzymes may have advantageous properties over existing nucleases. Detailed studies will then have to be performed to properly understand the design parameters of these novel CRISPR-Cas systems when they are deployed as genome and transcriptome engineering tools. Hence, while our protocol serves as a good reference for current genome editing efforts, it will need to be continuously updated with latest information about the new systems as they emerge.
The authors have nothing to disclose.
M.H.T. is supported by an Agency for Science Technology and Research’s Joint Council Office grant (1431AFG103), a National Medical Research Council grant (OFIRG/0017/2016), National Research Foundation grants (NRF2013-THE001-046 and NRF2013-THE001-093), a Ministry of Education Tier 1 grant (RG50/17 (S)), a startup grant from Nanyang Technological University, and funds for the International Genetically Engineering Machine (iGEM) competition from Nanyang Technological University.
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. |