Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole

Controlling protein expression using a degron is drawing more attention because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15–45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR–Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.


Introduction 41
Conditional depletion of a protein of interest (POI) is a powerful approach to 42 analyse its function in vivo, especially for POIs that are essential for cell viability. 43 Recently, conditional approaches using a degron have been drawing increased 44 attention [1]. A degron-fused protein can be rapidly and efficiently degraded when 45 needed, so that the primary defect arising from the depletion can be observed before 46 the phenotype is complicated or compromised by the secondary defects. For this 47 purpose, we pioneered the establishment of the auxin-inducible degron (AID) 48 technology to control degron-fused proteins in yeast and mammalian cells ( Figure  49 1A) [2]. When expressed in non-plant cells, TIR1 of rice (OsTIR1) forms a chimeric 50 SCF (Skp1-Cul1-F box) complex with the other endogenous components. The 51 SCF-OsTIR1 E3 ubiquitin ligase is only activated when IAA or NAA (a natural or 52 synthetic auxin, respectively) is bound ( Figure 1A). We identified a 7 kD degron 53 termed mini-AID (mAID) and others identified similar AID degrons [3][4][5]. A POI fused 54 with a mAID is recognized by SCF-OsTIR1 for ubiquitylation and subsequent 55 proteasomal degradation ( Figure 1A). 56 Recently, we showed that conditional human cells can be generated by 57 tagging endogenous genes with a mAID cassette using CRISPR-Cas9-based gene 58 tagging [6]. This technology has recently been applied in many studies. One 59 particular example is the assessment of chromosome architectures [7][8][9][10]. Moreover, 60 AID technology has been applied to other model organisms, such as fission yeast, 61 fruit fly, nematode, zebrafish and the parasitic Toxoplasma gondii [11][12][13][14][15]. These 62 studies support the idea that the AID technology can be used as a standard method 63 to achieve conditional protein depletion. However, a drawback of this technology is 64 that the expression level of mAID-fused proteins can be low in OsTIR1-expressing 4 cells [6]. This "basal degradation" might be caused by the presence of contaminating 66 auxin-like chemicals in bovine serum or culture media [1]. To achieve a tight control 67 of the expression of mAID-fused proteins, an improvement in this technology is 68 awaited. 69 Here, we describe a method to generate conditional human HCT116 and 70 DLD1 mutants by homology-directed repair (HDR)-mediated gene tagging using 71 CRISPR-Cas9 ( Figure 1B). We offer a new series of plasmids for N-or C-terminal 72 tagging with mAID and other tags. To overcome the problems associated with basal 73 degradation, we used a TIR1 inhibitor called auxinole ( Figure 5A) [16]. It was 74 possible to suppress basal degradation and rapidly recover expression after 75 depletion by supplementing culture media with auxinole. 76 77 2. Construction of CRISPR and donor plasmids for tagging 78 Figure 1B shows the procedures that were used to generate conditional AID 79 cells, which typically required one month of work. We previously reported mAID 80 tagging at the C terminus of a POI ( Figure 2B) [6]. We now developed a procedure 81 to tag a POI with mAID at the N terminus (Figure 2A) [17]. We offer N-or C-terminal 82 tagging plasmids with mAID or other tags at Addgene 83 (https://www.addgene.org/Masato_Kanemaki/) and the National Bio-resource Center 84 (NBRP) (http://dna.brc.riken.jp/en/gsb0000en/rdb08468) ( Figure 2C). 85

Construction of a CRISPR-Cas9 plasmid 86
To identify a CRISPR-Cas9 targeting site, we usually choose an appropriate 87 sequence within 50 bp upstream or downstream from the ATG or stop codon. We 88 use the following target finder sites. IDT custom Alt-R guide design: 89 https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM. WEG CRISPR 90 5 finder: https://www.sanger.ac.uk/htgt/wge/. We mainly use pX330-U6-Chimeric_BB-91 CBh-hSpCas9 (Addgene #42230) to express the SpCas9 nuclease and guide RNA 92 according to the protocol of Ran et al. [18]. However, it should be possible to use a 93 plasmid encoding other Cas9 variants. As discussed in the next section, it is 94 important to destroy or lose the target site within the genome by the HDR-mediated 95 insertion of a tagging cassette. 96

Construction of a donor plasmid 97
We described previously a method to construct donor plasmids by generating 98 homology arms (HAs) using long primers and gene synthesis [6]. A downside of this 99 strategy is that the HAs are relatively short (up to 200 bp each) and that it can be 100 costly. As an alternative (and economic) approach, we describe a method to clone 101 HAs from the genomic DNA (Figure 3). The N-or C-terminal coding region (about 102 1000 bp) is amplified by PCR, followed by cloning into a conventional cloning 103 plasmid (such as pBluescript II). After confirming the sequence, a cloning site for 104 inserting a tagging cassette with a selection marker is created by inverse PCR. The 105 cassette is cloned at the cloning site to complete the construction of the donor 106 plasmid, which contains the HA at both ends (about 500 bp each) ( Figure 3A and B). 107 Importantly, the donor plasmid has to be designed to destroy the CRISPR target site 108 when inserted into the genome. In the case of targeting a noncoding locus, it is 109 possible to delete part of the target sequence, unless a functional noncoding element 110 is absent. In the case of targeting a coding locus within a gene, it is important to 111 introduce silent mutations, to avoid re-cutting after HDR-mediated insertion. Another 112 important point is that a cassette encoding a tag has to be cloned in frame with the 113 gene of interest, to be able to express a fusion protein. All cloning procedures can be 114 carried out using a standard cloning method. In cases in which standard cloning 115 6 using restriction enzymes and ligase is difficult, it is possible to employ a 116 recombinase-mediated cloning method, such as In-Fusion cloning or Gibson 117 assembly. 118 To select clones with a biallelic insertion efficiently, we transfected two donor 119 plasmids containing a Neo or Hygro marker, respectively, for dual-antibiotic selection 120 [6]. As an alternative approach, it is possible to generate clones with a biallelic 121 insertion using a single donor plasmid with a single selection marker. In this case, 122 both monoallelic and biallelic clones can be obtained. 123 124

Transfection and colony formation 125
We established near-diploid colon cancer cell lines, HCT116 and DLD1, 126 constitutively or conditionally expressing OsTIR1 (CMV-or Tet-OsTIR1, respectively) 127 by inserting the transgene at the safe AAVS1 locus [6]. These parental cell lines are 128 available from NBRP or our laboratory upon request. Alternatively, it is possible to 129 generate a new parental cell line. In this case, plasmids that can be used for 130 introducing OsTIR1 at AAVS1 are available at Addgene and NBRP ( Figure 2D) [6]. 131 A CRISPR-Cas9 plasmid for targeting the AAVS1 locus is also available from 132 Addgene (AAVS1 T2 CRISPR in pX330: #72833).

Antibiotic selection and colony formation 152
The final concentration of antibiotics is shown below. One 96-well plate will be used to prepare a frozen stock and the other will be used to 185 prepare genomic DNA for PCR genotyping.

Genotyping by PCR 204
1. Design appropriate primers to check the insertion by PCR (Figure 4A and B). 205 We designed a primer set to detect both the wild-type (WT) and inserted alleles 206 antibodies are available to detect mAID and OsTIR1 (MBL M214-3 and PD048, 250 respectively). The example of mAID-fused CENPC is shown in Figure 4D.  Figure 5B). This showed that OsTIR1 289 expression, even in the absence of auxin, induced a mitotic phenotype that was 290 analogous to knockdown or inhibition of dynein [19,20]. The addition of auxinole 291 together with doxycycline clearly suppressed the downregulation of DHC1-mACl and 292 the mitotic arrest ( Figure 5B). To test whether DHC1-mACl could be rapidly depleted, 293 we added doxycycline with or without auxinole for 24 h. We monitored the 294 expression levels of DHC1-mACl by flow cytometry, and found that basal 295 degradation was mostly suppressed in the cells treated with doxycycline and 296 auxinole (Figure 5C, compare the boxes shown in red). Subsequently, the culture 297 media was replaced with fresh one containing doxycycline and IAA, but not auxinole. 298 Figure 5C shows that DHC1-mACl was rapidly degraded after medium replacement 299 and was mostly depleted within 4 h. 300 An advantage of AID technology is that the expression level of mAID-fused 301 proteins can be reversibly controlled [2]. We expected that auxinole would be useful 302 for re-expression after depletion, because IAA-bound OsTIR1 can remain active for a 303 while, even after the removal of IAA from the culture medium. To test this idea, we 304 used HCT116 CMV-OsTIR1 cells in which the cohesin subunit RAD21 was fused to 305 mAID-mClover (RAD21-mACl) [6]. Initially, we depleted RAD21-mACl by adding IAA 306 for 24 h ( Figure 6A). Subsequently, we replaced the medium with fresh media with 307 or without auxinole, and collected time-course samples to monitor the expression 308 levels of RAD21-mACl by flow cytometry (Figure 6B). We found that recovery of 309 RAD21-mADl was significantly rapid and sharp when auxinole was added, compared 310 with cells without auxinole. These results suggest that the OsTIR1 inhibitor auxinole 311 is useful for the tight control of the expression of mAID-fused proteins in human cells. 312 313

Conclusion 314
We described a CRISPR-Cas9-based method that can be used to fuse 315 endogenous POIs to mAID and other tags. We developed new plasmids for N-316 terminal tagging, so that it is now possible to tag the N and C termini of POIs ( Figure  317 2). To suppress basal degradation in cells expressing OsTIR1, we used the OsTIR1 318 antagonist auxinole (Figure 5A). Even in the Tet-OsTIR1 background cells, it is now 319 possible to induce rapid degradation of mAID-fused proteins by inducing OsTIR1 in 320 the presence of auxinole ( Figure 5C). Moreover, auxinole is useful for the re-321 expression of mAID-fused POIs after depletion ( Figure 6B). The use of auxinole 322 allows the rapid, tight and efficient control of the expression of mAID-fused POIs. 323 15 Other genetic systems also enable the control of degron-or tag-fused POIs using a 324 chemical ligand [1,[21][22][23]. However, to the best of our knowledge, there are no 325 inhibitors that allow the tight control of these systems. AID technology-now 326 combined with the degradation inducer, auxin, and the inhibitor, auxinole-will be 327 particularly useful to dissect biological networks, such as transcriptional cascades, 328 signal transduction and cell-cycle control systems, in which a primary defect caused 329 by the loss of a POI leads to secondary defects. We hope that the method described