Name: Genome Editing in Mammalian Cell Lines using CRISPR-Cas (Video) | JoVE Journal
Uploaded: 2019-04-11T12:30:00.000+00:00
Duration: 7 min 56 s
Description: Learn CRISPR-Cas9 genome editing to create knock-out and knock-in lines in mammalian cells. This efficient protocol enables precise genetic modifications for studying gene function and disease mechanisms.

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5 minutes

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.

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<li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-programmed+genome+editing+in+human+cells%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">RNA-programmed genome editing in human cells.</a> Elife. 2, e00471(2013).</li>
<li>Cho, S. W., Kim, S., Kim, J. M., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+genome+engineering+in+human+cells+with+the+Cas9+RNA-guided+endonuclease%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.</a> Nature Biotechnology. 31 (3), 230-232 (2013).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+evaluation+of+CRISPR-Cas+systems+reveals+design+principles+for+genome+editing+in+human+cells%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells.</a> Genome Biology. 19 (1), 62(2018).</li>
<li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+genome+editing+using+Staphylococcus+aureus+Cas9%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo genome editing using Staphylococcus aureus Cas9.</a> Nature. 520 (7546), 186-191 (2015).</li>
<li>Hou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+genome+engineering+in+human+pluripotent+stem+cells+using+Cas9+from+Neisseria+meningitidis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+U+S+A%22%5BJournal%5D)">Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.</a> Proceedings of the National Academy of Sciences U S A. 110 (39), 15644-15649 (2013).</li>
<li>Kim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+genome+editing+with+a+small+Cas9+orthologue+derived+from+Campylobacter+jejuni%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.</a> Nature Communications. 8, 14500(2017).</li>
<li>Edraki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Compact%2C+High-Accuracy+Cas9+with+a+Dinucleotide+PAM+for+In+Vivo+Genome+Editing%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.</a> Molecular Cell. , (2018).</li>
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<li>Boratyn, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLAST%3A+a+more+efficient+report+with+usability+improvements%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">BLAST: a more efficient report with usability improvements.</a> Nucleic Acids Research. 41 (Web Server issue), W29-W33 (2013).</li>
<li>Hsu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li>
<li>Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CHOPCHOP%3A+a+CRISPR%2FCas9+and+TALEN+web+tool+for+genome+editing%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.</a> Nucleic Acids Research. 42 (Web Server issue), W401-W407 (2014).</li>
<li>Heigwer, F., Kerr, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((E-CRISP%3A+fast+CRISPR+target+site+identification%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">E-CRISP: fast CRISPR target site identification.</a> Nature Methods. 11 (2), 122-123 (2014).</li>
<li>Haeussler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biology. 17 (1), 148(2016).</li>
<li>Bae, S., Park, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cas-OFFinder%3A+a+fast+and+versatile+algorithm+that+searches+for+potential+off-target+sites+of+Cas9+RNA-guided+endonucleases%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases.</a> Bioinformatics. 30 (10), 1473-1475 (2014).</li>
<li>Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., Corn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancing+homology-directed+genome+editing+by+catalytically+active+and+inactive+CRISPR-Cas9+using+asymmetric+donor+DNA%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.</a> Nature Biotechnology. 34 (3), 339-344 (2016).</li>
<li>Richardson, C. D., Ray, G. J., Bray, N. L., Corn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-homologous+DNA+increases+gene+disruption+efficiency+by+altering+DNA+repair+outcomes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.</a> Nature Communications. 7, 12463(2016).</li>
<li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li>
<li>Zhang, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+precise+knockin+with+a+double+cut+HDR+donor+after+CRISPR%2FCas9-mediated+double-stranded+DNA+cleavage%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.</a> Genome Biology. 18 (1), 35(2017).</li>
<li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+engineering+using+the+CRISPR-Cas9+system%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li>
<li>Shmakov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+and+evolution+of+class+2+CRISPR-Cas+systems%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Diversity and evolution of class 2 CRISPR-Cas systems.</a> Nature Reviews Microbiology. 15 (3), 169-182 (2017).</li>
<li>Moreno-Mateos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cpf1+mediates+efficient+homology-directed+repair+and+temperature-controlled+genome+editing%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.</a> Nature Communications. 8 (1), 2024(2017).</li>
<li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR%2FCas9+delivery%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> Elife. 3, e04766(2014).</li>
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</ol>

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,</su...

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.
<img alt="CRISPR gene editing process flowchart; sgRNAs design, cloning, cell transduction, clone screening." class="xfigimg" src="/files/ftp_upload/59086/59086fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: An overview of the workflow to generate genome edited human cell lines. <a href="https://www.jove.com/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

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 7</...

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

Research

JoVE Journal

Genetics

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.

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

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.

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

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—in essence, analyzing onsite mutagenesis.

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

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.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

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

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

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

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.

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

The protocol describes methods for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenome editing in human cell lines using plasmid DNA transfection and mRNA nucleofection.

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

The delivery of genome editors as RNAs is a particularly potent means of modifying the genomes of primary mammalian cells and cell lines. The goal of this protocol is to deliver genome editors as mRNA into primary mammalian cells and cell lines, followed by targeted Illumina sequencing and analysis to quantify editing outcomes.

Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA

Learn CRISPR-Cas9 genome editing to create knock-out and knock-in lines in mammalian cells. This efficient protocol enables precise genetic modifications for studying gene function and disease mechanisms.

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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