Name: genome editing in mammalian cell lines using crispr-cas
Uploaded: 2019-04-11T12:30:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

M.H.T. is supported by an Agency for Science Technology and Research&#8217;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.

The authors do not have competing financial interests.

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 &#8220;double-cut&#8221; 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 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&#8217; end of the protospacer, while the PAM of Cas12a (also called Cpf1) is at the 5&#8217; 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 &#8211; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59086/59086fig1.jpg" /> 
Figure 1: An overview of the workflow to generate genome edited human cell lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59086/59086fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>T4 Polynucleotide Kinase (PNK)</td><td>NEB</td><td>M0201</td><td></td></tr><tr><td>Shrimp Alkaline Phosphatase (rSAP)</td><td>NEB</td><td>M0371</td><td></td></tr><tr><td>Tris-Acetate-EDTA (TAE) Buffer, 50X</td><td>1st Base</td><td>BUF-3000-50X4L</td><td>Dilute to 1X before use. The 1X solution contains 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.</td></tr><tr><td>Tris-EDTA (TE) Buffer, 10X</td><td>1st Base</td><td>BUF-3020-10X4L</td><td>Dilute to 1X before use. The 1X solution contains 10 mM Tris (pH 8.0) and 1 mM EDTA.&amp;nbsp;</td></tr><tr><td>BbsI</td><td>NEB</td><td>R0539</td><td></td></tr><tr><td>BsmBI</td><td>NEB</td><td>R0580</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202</td><td>400,000 units/ml</td></tr><tr><td>Quick Ligation Kit</td><td>NEB</td><td>M2200</td><td>An alternative to T4 DNA Ligase.</td></tr><tr><td>Rapid DNA Ligation Kit</td><td>Thermo Scientific</td><td>K1423</td><td>An alternative to T4 DNA Ligase.</td></tr><tr><td>Zero Blunt TOPO PCR Cloning Kit</td><td>Thermo Scientific</td><td>451245</td><td>The salt solution comes with the TOPO vector in the kit.</td></tr><tr><td>NEBuilder HiFi DNA Assembly Master Mix</td><td>NEB</td><td>E2621L</td><td>Kit for Gibson assembly.</td></tr><tr><td>One Shot Stbl3 Chemically Competent E.Coli</td><td>Thermo Scientific</td><td>C737303</td><td></td></tr><tr><td>LB Broth (Lennox), powder</td><td>Sigma Aldrich</td><td>L3022</td><td>Reconstitute in ddH&lt;sub&gt;2&lt;/sub&gt;0, and autoclave before use</td></tr><tr><td>LB Broth with Agar (Lennox), powder</td><td>Sigma Aldrich</td><td>L2897</td><td>Reconstitute in ddH&lt;sub&gt;2&lt;/sub&gt;0, and autoclave before use</td></tr><tr><td>SOC media</td><td>-</td><td>-</td><td>2.5 mM KCl, 10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM glucose in 1 L of LB Broth</td></tr><tr><td>Ampicillin (Sodium), USP Grade</td><td>Gold Biotechnology</td><td>A-301</td><td></td></tr><tr><td>REDiant 2X PCR Mastermix</td><td>1st Base</td><td>BIO-5185</td><td></td></tr><tr><td>Agarose</td><td>1st Base</td><td>BIO-1000</td><td></td></tr><tr><td>T7 Endonuclease I</td><td>NEB</td><td>M0302</td><td></td></tr><tr><td>Plasmid DNA Extraction Miniprep Kit</td><td>Favorgen</td><td>FAPDE 300</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), High Glucose</td><td>Hyclone</td><td>SH30081.01</td><td>4.5 g/L Glucose, no L-glutamine, HEPES and Sodium Pyruvate</td></tr><tr><td>L-Glutamine, 200mM</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>Penicillin-Streptomycin, 10, 000U/mL</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>0.25% Trypsin-EDTA, 1X</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SV30160</td><td>FBS is heat inactivated before use at 56 &lt;sup&gt;o&lt;/sup&gt;C for 30 min</td></tr><tr><td>Phosphate Buffered Saline, 1X</td><td>Gibco</td><td>20012</td><td></td></tr><tr><td>jetPRIME transfection reagent</td><td>Polyplus Transfection</td><td>114-75</td><td></td></tr><tr><td>QuickExtract DNA Extraction Solution, 1.0</td><td>Epicentre</td><td>LUCG-QE09050</td><td></td></tr><tr><td>ISOLATE II Genomic DNA Kit</td><td>Bioline</td><td>BIO-52067</td><td>An alternative to QuickExtract</td></tr><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>6X DNA Loading Dye</td><td>Thermo Scientific</td><td>R0611</td><td>10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA</td></tr><tr><td>Protease Inhibitor Cocktail, Set3</td><td>Merck</td><td>539134</td><td></td></tr><tr><td>Nitrocellulose membrane, 0.2&amp;micro;m</td><td>Bio-Rad</td><td>1620112</td><td></td></tr><tr><td>Tris-glycine-SDS buffer, 10X</td><td>Bio-Rad</td><td>1610772</td><td>Dilute to 1X before use. The 1x solution contains 25 mM Tris, 192 mM glycine, and 0.1% SDS.</td></tr><tr><td>Tris-glycine buffer, 10X</td><td>1st base</td><td>BUF-2020</td><td>Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine.</td></tr><tr><td>Ponceau S solution</td><td>Sigma Aldrich</td><td>P7170</td><td></td></tr><tr><td>TBS, 20X</td><td>1st base</td><td>BUF-3030</td><td>Dilute to 1X before use. The 1x solution contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl.</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P9416</td><td></td></tr><tr><td>Skim Milk for immunoassay</td><td>Nacalai Tesque</td><td>31149-75</td><td></td></tr><tr><td>WesternBright Sirius-femtogram HRP</td><td>Advansta</td><td>K12043</td><td></td></tr><tr><td>Antibody for &amp;beta;-actin (C4)</td><td>Santa Cruz Biotechnology</td><td>sc-47778</td><td>Lot number: C0916</td></tr><tr><td>MiSeq system</td><td>Illumina</td><td>SY-410-1003</td><td></td></tr><tr><td>NanoDrop spectrophotometer</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Thermo Scientific</td><td>Q33226</td><td></td></tr><tr><td>EVOS FL Cell Imaging System</td><td>Thermo Scientific</td><td>AMF4300</td><td></td></tr><tr><td>CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458)</td><td>Addgene</td><td>48138</td><td>Single vector system: The gRNA is expressed from the same plasmid.</td></tr><tr><td>CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA</td><td>Addgene</td><td>61591</td><td>Single vector system: The gRNA is expressed from the same plasmid.</td></tr><tr><td>CRISPR plasmid: xCas9 3.7</td><td>Addgene</td><td>108379</td><td>Dual vector system: The gRNA is expressed from a different plasmid.</td></tr><tr><td>CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9</td><td>Addgene</td><td>42230</td><td>Single vector system: The gRNA is expressed from the same plasmid.</td></tr><tr><td>CRISPR plasmid: hCas9</td><td>Addgene</td><td>41815</td><td>Dual vector system: The gRNA is expressed from a different plasmid.</td></tr><tr><td>CRISPR plasmid: eSpCas9(1.1)</td><td>Addgene</td><td>71814</td><td>Single vector system: The gRNA is expressed from the same plasmid.</td></tr><tr><td>CRISPR plasmid: VP12 (SpCas9-HF1)</td><td>Addgene</td><td>72247</td><td>Dual vector system: The gRNA is expressed from a different plasmid.</td></tr></tbody></table>

genome editing in mammalian cell lines using crispr-cas

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.

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.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59086/59086fig7.jpg" /> 
Figure 7: Cloning of sgRNA-expressing CRISPR plasmids. (a) Image showing digested and undigested plasmids after gel electrophoresis. Lanes 1&#8210;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59086/59086fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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&#8210;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.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59086/59086fig8.jpg" /> 
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 &#181;m. (b) Representative FACS plots. Transfected cells (right panel) are gated against a non-transfected control (left panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59086/59086fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59086/59086fig9.jpg" /> 
Figure 9: Evaluating the extent of genome editing. (a) Representative gel image showing the results of a T7E1 assay. Lanes 1&#8210;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 &#34;-&#34;. (b) Representative gel image showing the results of a RFLP assay. Lanes 1&#8210;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59086/59086fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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).
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/59086/59086fig10.jpg" /> 
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. &#8220;WT&#8221; stands for wildtype cells, where GLUL is highly expressed. ACTB (&#946;-actin) serves as a loading control (see the Table of Materials). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59086/59086fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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 ...

genome-editing-in-mammalian-cell-lines-using-crispr-cas

Nanyang Technological University

Research

JoVE Journal

Genetics

21.4K Views.  Agency for Science Technology and Research. Dit protocol maakt gebruik van CRISPR-Cas9 om knock-out- of knock-inlijnen te maken. Het belangrijkste voordeel van deze techniek is dat het eenvoudig en efficiënt te volgen vooral voor beginnende onderzoekers. Voor celdoorgang behandel u 's nachts gekweekte cellen met twee milliliters van 0,25% trypsine EDTA per plaat bij 37 graden Celsius gedurende twee minuten.Wanneer de cellen van de plaatbodems zijn opgeheven, neutraliseert u de enzymatische reactie met twee milliliter celkweekme...

Video: Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Developmental Biology

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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