Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection
© Estep et al. 2016
Received: 17 September 2015
Accepted: 18 March 2016
Published: 2 April 2016
Targeted genomic editing using the CRISPR/Cas9 methodology has opened exciting new avenues in probing gene function in virtually any model system, including cultured mammalian cells. Depending on the desired mutation, several experimental options exist in the isolation of clonal lines, such as selection with introduced markers, or screening by PCR amplification of genomic DNA. However, streamlined approaches to establishing deletion and tagging mutants with minimal genomic perturbation are of interest in applying this methodology.
We developed a procedure for rapid screening of clonal cell lines for the deletion of a protein of interest following CRISPR/Cas9 targeting in the absence of selective pressure based on dot immunoblots. To assess the technique, we probed clonal isolates of 293-TREx cells that were targeted with three separate sgRNAs against the HuR gene. Validation of knockout candidates by western blot indicated that the normalized protein abundances indicated by the dot blot serve as accurate predictors of deletion. In total, 32 independent biallelic deletion lines out of 248 screened clones were isolated, and recovery of null mutants ranged from 6 to 36 % for the individual sgRNAs. Genomic sequencing verified small deletions at the targeted locus.
Clonal screening for CRISPR/Cas9-mediated editing events using dot immunoblot is a straightforward and efficient approach that facilitates rapid generation of genomic mutants to study gene function.
KeywordsCRISPR/Cas9 Clonal selection Screening Dot blot
Manipulating protein levels and activities is a principal tool in understanding the functions and the relationships of these molecular components in cells. Transient perturbations involve chemical inhibition (and activation) with small molecules, overexpression from non-integrating vectors, or knockdown by RNA interference . Often more experimentally desirable, stable genetic alteration can be achieved by integration of overexpression and shRNA constructs , or by genomic editing of the endogenous protein locus with transcription activator-like effector nucleases (TALENs) , zinc-finger nucleases [4, 5], and, more recently, by RNA-guided nucleases based on the clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 system [6–11]. Combinations of stable integration and modification with temporal control include the use of chemically inducible systems, such as tetracycline repression/activation [12, 13], tamoxifen control of Cre-ER recombination [14, 15], and more modular control of expression, localization and activity by small molecules [16–18].
In the targeted modification strategies, the researcher has the option to isolate correct clones by introducing a selectable marker at the genomic locus. While this approach is beneficial if the sgRNA has low efficiency, or if the mutation confers a strong selective disadvantage to the cells, it requires the construction of repair template vectors containing the marker and homology arms specific to the locus . Furthermore, it leaves a significantly-sized, transcribed insertion that may affect the expression of nearby genes, although it may be excised through an additional Cre-mediated step, if surrounded by loxP sites .
However, the relatively high efficiency of Cas9 targeting allows for clonal screening without selection. In a common approach, purification of genomic DNA from isolated clones permits PCR amplification followed by sequencing, deletion analysis or restriction digest . In the present study, we describe a simple method for isolation of clonal deletion mutants based on CRISPR/Cas9 targeting combined with dot immunoblot analysis and validated by western blots and DNA sequencing. Screening directly for protein production ensures proper knockout, avoiding signal from in-frame deletions/mutations that may not eliminate its function. The approach can also be applied to screening for insertions of tags mediated by homologous recombination.
Results and discussion
We wished to apply the CRISPR/Cas system to establish stable cell lines completely lacking specific protein function. As with other genetic mutant approaches, it is beneficial to isolate several independently derived knockout clones to control for secondary mutations. In the case of CRISPR/Cas, off-target cleavage events at sites of imperfect sgRNA complementarity are known to occur [29, 30], and the use of different sgRNAs can mitigate the possibility of spuriously observed phenotypes. We used pre-designed human genomic target sites for the S. pyogenes Cas9 effector nuclease, picking the reduced set of three sites closer to the 5′ end of the gene . The targeted sites were introduced into sgRNA-producing context in a plasmid that also co-expresses the Cas9 protein, pSpCas9(BB) [24, 28].
We applied the above strategy to generate a knockout of the HuR (ELAVL1) protein in 293-TREx cells, targeting exons 3, 4, and 5 with three distinct sgRNAs. Most of the expanded clones exhibited parental growth rates and morphology, while several lines grew substantially slower and/or with altered cell shape. However, these features did not correlate with the absence of HuR (see below), likely reflecting spurious mutations acquired due to CRISPR/Cas off-targeting or simply during the procedure. Thus, HuR is dispensable to normal growth rates under optimal conditions in 293-TREx cells; however, if the protein of interest is expected to affect the division or morphology of cells, it can form the basis of a phenotypic pre-screen during clonal expansion. Mutations that are substantially detrimental to growth rates can still be recovered, since the clonal isolation begins soon after the introduction of the sgRNA/Cas9.
Number of analyzed and validated KO clonal lines for the sgRNAs used
# of clones tested by dot blot
# of validated KOs
KO percentage (%)
Aside from targeting individual genes for knockout, the CRISPR/Cas system allows many additional means of genome editing. We assessed whether our screening protocol can be used to achieve simultaneous knockout of tandemly arranged genes by inducing large chromosomal deletions between two sgRNA target sites. Small deletions have been previously achieved without selection , while larger biallelic and monoallelic deletions have been created after sorting for strong Cas9 expression , or in haploid embryonic stem cells . In our hands, attempts to excise the ~250 Kbp human Ago4, 1, 3 locus by targeting Ago4 and Ago3 while blotting for Ago1 have not yielded any complete deletions (data not shown). Thus, elimination of large genomic fragments may require the use of homologous recombination templates spanning the deletion, enrichment for cells expressing Cas9 (without genomic insertion), and/or incorporation of selectable markers at the deletion site. Similarly, gene mutagenesis without deletion would require homologous templates involving selection or protein tagging. In these cases, dot blotting for the tag may still be used as a screening step. It should be pointed out that directly blotting for the protein under study is limited by antibody availability, and assaying of secreted proteins may necessitate the collection of culture media instead of cell pellets.
The CRISPR/Cas system is transforming modern genetics and molecular biology by offering unprecedented ways of genomic manipulation. We have described a methodology to isolate simple deletion mutants of a gene of interest by CRISPR/Cas based on dot-blot screening for the resulting protein product, without the need for intervening selection steps. The advantages of the method lie in the ease of upfront preparation (only the sgRNA constructs need to be created, based on short synthetic oligonucleotides), the relative simplicity of the screening procedure, and the minimal genomic perturbations to the resulting clonal lines. For a typical knockout experiment, our estimates indicate a time saving of 4–5 days relative to direct western blotting, or on the order of weeks relative to design and construction of selection steps. The protocol can be modified to allow for editing with homologous recombination templates, by blot screening for protein tags introduced on the template. The presented techniques should expand the available toolkit for the application of biochemistry and molecular and cell biology approaches to the study of protein function in mammalian cell culture.
Preparation of Cas9-sgRNA plasmids
The pSpCas9(BB) plasmid was a gift from Feng Zhang (Addgene plasmid #42230). Single guide RNA sequences were constructed to target the pre-designed loci in the third, forth, and fifth exon of the HuR gene (HuR-3, TGTGAACTACGTGACCGCGA;HuR-4,CGGGCGAGCATACGACACCT;HuR-5,CCGGATAAACGCAACCCCTC) and cloned into the parental plasmid as previously described . Clones were tested for correct inserts using Sanger sequencing.
Cell Culture, transfections, and dilutions
293-TREx cells were grown in Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose, l-glutamine and sodium pyruvate (Corning Cellgro Ref:10-013-CV) that was supplemented with 10 % Fetal Bovine Serum (HyClone FBS Characterized Cat: SH30071.03) and 10 units/ml of Penicillin–Streptomycin (HyClone Cat: SV30010). Cultured plates were grown in a humidified incubator supplying 5 % CO2 at 37 °C.
Two independent transfection events were performed for each construct. First, cells were grown in a 6-well format to approximately 60 % confluency and transfected with 2.5 µg of the prepared Cas9-sgRNA plasmid using the calcium phosphate method. 24 h later cells were passaged into 10 cm plates. 24 h following passaging, cells were again transfected with 12.5 µg plasmid using the TransIT-LT1 reagent (Mirus, Cat: MIR 2300). Cells were allowed to reach 100 % confluency before seeding out into individual clones. After the transfections, efficiency was estimated to be 70–80 % by GFP expression in a parallel transfection with a modified version of the pMSCV-PIG vector.
To isolate individual clones, cells were seeded at a density of 3–5 cells per 96-well. Colony growth in less that 60 % of the wells was observed over 3–4 weeks, and media was changed weekly to prevent drying of wells. Clone colonies that were visible to the eye were picked into 96-well plates by aspirating the media, dispensing 2 µl of fresh media onto the colony, scraping across the colony with the pipette tip, and drawing the media back. 24–48 h later, isolated clones that were growing as a clump were reseeded by pipette mixing in the well to encourage monolayer cell growth.
Dot blot and western blot
Individual clones were grown until 100 % confluency and collected for dot blot analysis, dislodging the monolayer of cells by pipeting within the well. 90 µl of the 100 µl total volume was then removed, spun down, and the cell pellet lysed in 10 µl of 1× Passive Lysis Buffer (Promega’s Dual-Luciferase Reporter Assay System, Cat: E1910). The remainder of the cells were propagated. 1 µl of cell lysate was pipetted onto dry nitrocellulose membrane (Bio-Rad, Cat: 162-0115) to form a dot. Each sample was blotted twice on two separate membranes, creating two identical patterns of samples, blocked in 5 % milk in TBST for 1 h at room temperature, and blotted for HuR (Santa Cruz Biotechnology, clone 3A2, Cat: sc-5261, 1:1000–1:5000 dilution) and Pum2 (Bethyl Labs, Cat: A300-202A, 1:1000 dilution) in 5 % milk in TBST for 1 h, with 3 × 5 min TBST washes in between incubation steps. Anti-Mouse-HRP-conjugated secondary antibody was from Cell Signaling (anti-mouse, Cat: 7076). Anti-Rabbit-HRP-Conjugated Secondary Antibody was from Cell Signaling (anti-rabbit, Cat: 7074). Blotted membranes were imaged on a Biorad ChemiDoc MP imager and quantified by Image Lab: identical circles were centered on each dot, and the signal volume was computed by the software. Local background values for each dot were computed and subtracted by Image Lab. Western blotting was performed as described above.
Sequencing of genomic DNA
To amplify a genomic fragment flanking the HuR-5 target region, primers were designed containing BbsI restriction sites with distinct overhangs (HuR-5 For, CGACTTGAAGACCTCACCTGTGATGGGCTCAGAGGAACC; HuR-5 Rev, CGACTTGAAGACCTAAACAGACTTCTAGCCTGGCCCAC). Genomic DNA of the parental 293-TREx line and the HuR-5.B5 mutant was isolated by standard SDS/Proteinase K lysis followed by phenol/chloroform extraction and isopropanol precipitation. The target region was PCR amplified using Q5 High Fidelity DNA Polymerase (New England Biolabs, Cat:M0491S), and cloned by Golden Gate cloning into a modified pRL-TK plasmid bearing two consecutive BbsI sites (ThermoFisher Scientific Cat# FERFD1014). Isolated bacterial colonies were mini-prepped and Sanger sequenced.
clustered, regularly interspaced short palindromic repeats
single guide RNA
short hairpin RNA
transcription activator-like effector nuclease
non-homologous end joining
FVK designed the project; ELS and GAS designed and cloned the sgRNA/Cas9 constructs; JAE performed the transfection, clone expansion, dot blotting, western blot, sequencing validation and analyzed data; FVK and JAE wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Megan Lee and Alyssa Rodriguez for help with the experiments, the IIGB sequencing core for technical assistance, and Feng Zhang for sharing reagents.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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