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 border="0" cellpadding="0" cellspacing="0" style="width:1424px;" width="1424">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 Polynucleotide Kinase (PNK)</td>
			<td style="width:175px;">NEB</td>
			<td style="width:119px;">M0201</td>
			<td style="width:688px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Shrimp Alkaline Phosphatase (rSAP)</td>
			<td>NEB</td>
			<td style="width:119px;">M0371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">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 height="21">
			<td height="21" style="height:21px;width:443px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BbsI</td>
			<td>NEB</td>
			<td>R0539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BsmBI</td>
			<td>NEB</td>
			<td>R0580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 DNA Ligase</td>
			<td>NEB</td>
			<td style="width:119px;">M0202</td>
			<td>400,000 units/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Quick Ligation Kit</td>
			<td>NEB</td>
			<td style="width:119px;">M2200</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Rapid DNA Ligation Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">K1423</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Zero Blunt TOPO PCR Cloning Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">451245</td>
			<td>The salt solution comes with the TOPO vector in the kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>NEB</td>
			<td style="width:119px;">E2621L</td>
			<td>Kit for Gibson assembly.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">One Shot Stbl3 Chemically Competent E.Coli</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth with Agar (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L2897</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">SOC media</td>
			<td>-</td>
			<td style="width:119px;">-</td>
			<td>2.5 mM KCl, 10 mM MgCl2, 20 mM glucose in 1 L of LB Broth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ampicillin (Sodium), USP Grade</td>
			<td>Gold Biotechnology</td>
			<td style="width:119px;">A-301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">REDiant 2X PCR Mastermix</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-5185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Agarose</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T7 Endonuclease I</td>
			<td>NEB</td>
			<td style="width:119px;">M0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Plasmid DNA Extraction Miniprep Kit</td>
			<td>Favorgen</td>
			<td style="width:119px;">FAPDE 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Dulbecco&#39;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 height="21">
			<td height="21" style="height:21px;width:443px;">L-Glutamine, 200mM</td>
			<td>Gibco</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Penicillin-Streptomycin, 10, 000U/mL</td>
			<td>Gibco</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">0.25% Trypsin-EDTA, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:443px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td style="width:119px;">SV30160</td>
			<td>FBS is heat inactivated before use at 56 oC for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Phosphate Buffered Saline, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">20012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">jetPRIME transfection reagent</td>
			<td>Polyplus Transfection</td>
			<td style="width:119px;">114-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">QuickExtract DNA Extraction Solution, 1.0</td>
			<td>Epicentre</td>
			<td style="width:119px;">LUCG-QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">ISOLATE II Genomic DNA Kit</td>
			<td>Bioline</td>
			<td style="width:119px;">BIO-52067</td>
			<td>An alternative to QuickExtract</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Q5 High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td style="width:119px;">M0491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Deoxynucleotide (dNTP) Solution Mix</td>
			<td>NEB</td>
			<td style="width:119px;">N0447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">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 height="21">
			<td height="21" style="height:21px;width:443px;">Protease Inhibitor Cocktail, Set3</td>
			<td>Merck</td>
			<td style="width:119px;">539134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Nitrocellulose membrane, 0.2&#181;m</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">1620112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine-SDS buffer, 10X</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine buffer, 10X</td>
			<td>1st base</td>
			<td style="width:119px;">BUF-2020</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ponceau S solution</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P7170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">TBS, 20X</td>
			<td>1st base</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:443px;">Tween 20</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Skim Milk for immunoassay</td>
			<td>Nacalai Tesque</td>
			<td style="width:119px;">31149-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">WesternBright Sirius-femtogram HRP</td>
			<td>Advansta</td>
			<td style="width:119px;">K12043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Antibody for &#946;-actin (C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-47778</td>
			<td>Lot number: C0916</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">MiSeq system</td>
			<td>Illumina</td>
			<td style="width:119px;">SY-410-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Qubit fluorometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">EVOS FL Cell Imaging System</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">AMF4300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458)</td>
			<td>Addgene</td>
			<td style="width:119px;">48138</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA</td>
			<td>Addgene</td>
			<td style="width:119px;">61591</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: xCas9 3.7</td>
			<td>Addgene</td>
			<td style="width:119px;">108379</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">42230</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: hCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">41815</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: eSpCas9(1.1)</td>
			<td>Addgene</td>
			<td style="width:119px;">71814</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: VP12 (SpCas9-HF1)</td>
			<td>Addgene</td>
			<td style="width:119px;">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

21.3K Views.  Agency for Science Technology and Research. 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.

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

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

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 ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

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

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

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 (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

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