Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

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1 1 Supplementary Materials Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange Authors William Kelton 1, Ann Cathrin Waindok 1, Theresa Pesch 1, Mark Pogson 1, Kyle Ford 1, Cristina Parola 1 & Sai T. Reddy 1, * Affiliations 1 Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland *Corresponding author. sai.reddy@ethz.ch 11 1 Supplementary Table 1: Plasmids and primers used in this work. Name Relevant characteristics or sequence (5 to 3 ) Source px458 Expression of Cas-9 nuclease and guide RNAs [Zhang], Addgene px458-g Expression of Cas-9 nuclease and guide RNA # This study px458-g3 Expression of Cas-9 nuclease and guide RNA #3 This study px458-g4 Expression of Cas-9 nuclease and guide RNA #4 This study px458-g7 Expression of Cas-9 nuclease and guide RNA #7 This study pmp Expression of Cas-9 nuclease and muliple guide RNAs This study pmp-g/4 Expression of Cas-9 nuclease and guide RNAs # and #4 This study MCS1 Minicircle production vector System Biosciences puc19 Bacterial cloning vector NEB, USA puc19- Storage vector containing the allele This study puc19-h-kd Storage vector containing the H-Kd allele This study ABC Mincircle storage vector for production of 'ABC' minicircle This study ABC Mincircle storage vector for production of self-cleaving 'ABC' minicircle This study ADE Mincircle storage vector for production of 'ADE' minicircle This study ADE Mincircle storage vector for production of self-cleaving 'ADE' minicircle This study p1 (WK46) GAACTCAGAAGTCGCTAATCGC p (WK47) CCCTGTAATACTCTGCATCACC p3 (WK5) CAGGGGATGGAACCTTCCAGAAGTG 1

2 13 p4 (WK4) CAGCCCTAGGTCAAGATGATAACAATCAAGG p5 (WK) GCTGGTGATGCAGAGTATTACAG p6 (WK3) GGTGACATCAACTTGAGATCTGGG p7 (WK) GCTGGTGAAGCAGAGAGACTCAG p8 (WK1) GGTGACTTTATCTTCAGGTCTGCT p9 (WK89) GGTCCGTGTGGGGCTTGCAG p1 (WK34) GGTAGGCCCTGAGTCTCTC p11 (WK11) GAACCAATCAGTGTCGCCGCG p1 (WK1) TAGTGACCCAGATTCTGGAAGTTTATTCATCTATC p13 (WK4) GATAGATGAATAAACTTCCAGAATCTGGGTCACTATTAAGCCAG CCCCGACACCC p14 (WK5) CGCGGCGACACTGATTGGTTCCACATTAATTGCGTTGCGCTCAC TG p15 (WK) GGCGGTGACGAAATACCTCAGCG p16 (WK8) TCCCTCACCTCATCAGCTCA p17 (WK7) AATGGACAGTGATGGTGGGC p18 (WK3) CCTTGATTGTTATCATCTTGACCTAGGGCTG p19 (WK369) /5'phos/CACCGATTTCGTCACCGCCGTGTCC p (WK37) /5'phos/AAACGGACACGGCGGTGACGAAATC p1 (WK375) /5'phos/CACCGCGTCACAGCCGAACATCCGC p (WK376) /5'phos/AAACGCGGATGTTCGGCTGTGACGC p3 (WK371) /5'phos/CACCGTCCTGTCCCCACAACTAACC p4 (WK37) /5'phos/AAACGGTTAGTTGTGGGGACAGGAC p5 (WK373) /5'phos/CACCGCTGTCTCTCCCAGATGGTAA p6 (WK374) /5'phos/AAACTTACCATCTGGGAGAGACAGC p7 (WK48) CTGCTCGGCTACTACAACC p8 (WK49) GATCAGAGGTCTGGGAGC p9 (WK343) GGACGCTGGCTATAAAGTCCAC p3 (WK43) GGTTGTAGTAGCCGAGCAG p31 (WK431) GCTCCCAGACCTCTGATC p3 (WK43) GGCTGTGGAAGGGAAGAC p33 (WK7) CTCTAGAGTCGACCCATGGG p34 (WK15) CAGATCTGAATTCACTAGTCGCG p35 (WK459) GTCTTCCCTTCCACAGCCCCTTTACCATCTGGGAGAGACAGCTC TAGAGTCGACCCATGGG p36 (WK46) GTGGACTTTATAGCCAGCGTCCCCGGGACACGGCGGTGACGAA ATCAGATCTGAATTCACTAGTCGCG p37 (WK116) GGTACCCAAGTTTCAGAGGAGAGAAAC p38 (WK13) CGTTTGTGAAGCTGCCTAACCCCAACAG p39 (WK19) GTTTCTCTCCTCTGAAACTTGGGTACCCACATTAATTGCGTTGCG CTCACTG p4 (WK13) CCCTGAACCCCTCTTATTAGATGTTGTGATTAAGCCAGCCCCGA CACCC p41 (WK17) GCAGTCCTGTGCTGAGGGGAC p4 (WK6) GCCTGGGAGAGGCCATGGC p43 (WK3) CTCTCACAGCTTTTCTTCTCACAGG p44 (WK159) GGTCCGTGTGGGGCT p45 (WK17) CCTGGAACTCACTTTGTAGACCAGG p46 (WK171) CCTTTAATCCCAGCACTCGGGAG

3 a Exon Nucleotide Differences H-Kd 4/64 8/7 3/76 19/76 9/117 /33 /3 1/39 1 bp Higher Identity Lower Identity b 5 homology CRISPR site T A T T T C G T C A C C G C C G T G T C C C G A C C C G G C C T C G G G Y F V T A V S R P G L G Exon H-Kd grna1 PAM c 3 homology CRISPR site T C T C T C C T T A C C C G A T G G T A A A G G T G A C A C T C T A G G L S L P D G K G D T L G Exon 7 H-Kd grna13 PAM d % of Max RAW64.7 % of Max RAW64.7 Count 3 1 SIINFEKL Conc 1 fm 1 nm 1 nm 1 nm nm Count 3 1 RAW64.7 SIINFEKL Conc 1 nm nm anti- anti- (488 nm) e Cell Line Cell Line Cell Line Supplementary Figure 1: Design of donor templates and selection of modified RAW64.7 macrophages. (a) Comparison of exon sequence identity at the nucleotide level for murine and H-Kd MHC alleles. (b) The 5 homology junction with the allele; the PAM recognition site in the donor template was altered from NGG > NGA to prevent cleavage by Cas9. (c) The 3 homology junction with the allele; 5 non-coding mutations were introduced to alter the grna13 recognition site to prevent cleavage by Cas9. (d) In order to capture modified cells without the use of integrating antibiotic or fluorescent selection markers, we evaluated the selectivity of monoclonal antibodies against the H-Kd and alleles expressed on the cell surface of RAW64.7 and cells. Robust separation of the two cell lines was

4 achieved for the H-Kd binding clone SF (PerCP-efluor 71, 1.5 µg/ml) but was not as pronounced for the binding clone AF (FITC, 5 µg/ml). We therefore titrated the amount of SIINFEKL peptide required for detection by the 5-D1.16 antibody (APC, 1.5 µg/ml) clone on cells expressing and found high discrimination when 1 nm of peptide was supplied. At this peptide concentration only minimal signal was detected on H-Kd positive RAW64.7 cells. (e) Additional representative FACS profiles for RAW64.7 clones isolated for expression following labeling for H-Kd and bound with SIINFEKL. 4

5 a RAW64.7 CD CD86 H-Kd CD8 5 CD b 1. Absorbance A Absorbance A B3Z - SIINFEKL SIINFEKL 64.7 B3Z - SIINFEKL SIINFEKL Supplementary Figure : B3Z T cell activation assay (a) Fold upregulation of antigen presenting cell markers CD8, CD86, and /d upon stimulation by LPS and/or IL-4 cytokine for RAW64.7 and immortalized cell lines as determined by FACS analysis. Cells were stained with 1.5 µg/ml anti-h- Kd (RAW64.7) or 5 µg/ml anti- () as well as 1.5 µg/ml anti-mouse CD86 and.3 µg/ml antimouse CD8. (b) Additional independent experiments of B3Z T cell hybridoma activation by + RAW64.7 cells. Each cell line was tested with and without SIINFEKL peptide and the absorbance at 57 nm was measured after 3 hours of incubation (N = 3). Error bars indicate 95% confidence intervals. 38 5

6 a c Cell line 9 Ø * 3* 9 *mutations shared between lines # shared # coding 3 1 b Minicircle (Self-linearizing) Minicircle containing EcoRI site C CT T C CA CA GC CC T C T A GA GT CGAC CC AT GGGGGC CC GC CC CA AC T GGGGT AA CC T T T GGGCT CC CC GGGC GC GA CT AGT GAA T T CA GA T C T GGGAC GC T GGC T A Arm grna13 grna1 AttRAttR EcoRI EcoRI Arm C CT T C CA CA GC CC CT T T AC CA T C T GGGAGAGAC AGCT CT AGAGT C GA CC CA T GGGGGCC CGCC CC AA CT GGGGT A AC CT T T GGGC T C CC CGGGCGCGAC T A GT GA AT T C AGAT CT GA T T T C GT CA CC GC CGT GT C CC GGGGAC GC T GGC T A Arm PAM grna13 AttR grna1 PAM Arm EcoRI d Knockout Control Linear Minicircle Minicircle (Self-linearizing) e bp 6 3 Linear Mincircle EcoRI Minicircle 1..1 Minicircle (S-L) EcoRI MC f bp 3 1 integration (p9/p1) RAW64.7 Linear Minicircle Minicircle (S-L) EcoRI MC Plasmid Plasmid

7 Supplementary Figure 3: Donor template sequencing and optimization for improved exchange efficiency. (a) The H-K1 locus was amplified in all cell lines by split pool PCR and three clones were Sanger sequenced from each line. Mutations were counted in the exchanged sequence that differ from the expected donor template sequence (Ø), that are shared between the sequenced clones, and that lead to coding changes on the amino acid level. The asterisk (*) indicates identical mutations were found in the clones suggesting a common progenitor. (b) Expected pmc-bespx-et1 minicircle sequence following recombination of AttB and AttP sites. An AttR site is generated and a unique EcoRI restriction enzyme site remains to allow for linearization of the construct. (c) Expected pmc-bespx-et minicircle sequence following recombination showing the layout of grna1 and grna13 sites that allow for self-linearizing upon expression of Cas9 in the cell. (d) Representative FACS data for percent allele exchange for each donor template. (e) Representative agarose gel of donor templates investigated for improved efficiency normalized to 1 µg/µl. Linear fragments were generated by PCR from plasmid templates ET1 (Linear). Minicircle DNA was prepared from both ET1 (Minicircle) and ET (Minicircle Self-linearizing (S-L)). EcoRI restriction enzyme was used to create linear DNA from the ET1 minicircle template (EcoRI MC). Plasmid DNA was uninduced ET1 that contained the bacterial backbone (Plasmid). (f) Primers p9 and p1 were used to assay correct integration of the various forms of donor templates at the correct H-K1 locus. Cells were sorted for GFP expression before analysis. 7