Using CRISPR-CAS9 for rapid fusion detection

Dr Mike Hubank Head of Clinical Genomics (Research), Royal Marsden Hospital, London Reader in Translational Genomics (ICR) Scientific Director North London GLH

Festival of Genomics, Jan 2020 Disclosures

• Personal or Laboratory support received from:

Astra Zeneca, Boehringer Ingelheim, Roche Diagnostics, Bristol Myers Squibb, Guardant Health, Eli Lilley. Acknowledgements

• Collaboration with DeepSeq, Nottingham University:

Royal Marsden University of Nottingham Lewis Gallagher Nadine Holmes Sara Ribeiro Victoria Wright Dorte Wren Matt Loose Suzanne McMahon Mike Hubank

Oxford Nanopore Michelle Hiscutt Gene Fusions of Clinical Significance

Found in many tumours including:

Sarcoma Diagnostic/Prognostic/Targetable Lymphoma Ependymoma e.g. PML-RARA – Acute Promyelocytic Acute Lymphoblastic Leukemia < 48 hr Acute Myeloid Leukemia ALK – partner (eg NPM1-ALK) Lorlatinib Glioma NTRK-partner Larotrectinib Medulloblastoma Astrocytoma Neuroblastoma Histiocytoma Currently Measured by : FISH; IHC eg BCR-ABL RT-PCR

Quick and relatively cheap, but need to know a likely positive. Not comprehensive. Fusions by RT-PCR 165 (ish) Gene Fusions on NHSE test directory

TGFBR3- ABL1 CIC-FOXO4 EWSR1-FLI1 JAK2 PAX3-FOXO1 MGEA5 ABL2 CIC-NUTM1 EWSR1-NR4A3 JAZF1-PHF1 PAX3-FOXO1A TLX1-TRD ALK CIC-NUTM2A EWSR1-PBX3 JAZF1-SUZ12 PAX3-NCOA1 TRB-TAL1 ALK-NPM cMYC EWSR1-POU5F1 KMT2A PAX7-FOXO1 TRD-LMO1 API2-MALT1 COL1A1-PDGFB EWSR1-WT1 KMT2A-AFF1 PAX7-FOXO1A TPM3-ALK ATF1-EWSR1 CREB1-EWSR1 EWSR1-ZNF444 KMT2A-MLLT1 PCM1-JAK2 TPM4-ALK ATF1-FUS CRLF2 FGFR1 MAML3-BCOR PDGFRA TRB-TAL1 BCL11B-TLX3 CSF1R FIP1L1-PDGFRA MAML3-PAX3 PDGFRB TRD-LMO1

BCL2 DDIT3-EWSR1 FLT3 MN1 PML-RARA TTYH1-C19MC BCL6 DDIT3-TLS FN1-FGF1 MYB-NFIB PVT1- USP6 WWTR1- BCOR DNAJB1-PRKACA FN1-FGFR1 MYB-QKI PVT1-MYC CAMTA1

BCOR-CCNB3 ELM4-ALK FUS-ATF1 MYBL1 RAF1 -NF1A YAP-C11orf95 YAP1- BCOR-CCNB3 EPC1-PHF1 FUS-CREB3L1 MYC RAF1 -SRGAP3 C11orf95 BCOR-MAML3 EPOR FUS-CREB3L2 MYH9 RET YAP1-TFE3 BCR-ABL ETV6-NTRK3 FUS-DDIT3 MYH9-USP6 ROS1 YAP1-TFE3

BRAF-AGK ETV6-PDGFRB FUS-ERG NAB2-STAT6 RUNX1-RUNX1t1 ZC3H7B-BCOR BRAF-AKAP9 ETV6-RUNX1 FUS-ERG NTRK-ETV6 SERPINE1-FOSB BRAF-CCDC6 EWSR1-ATF1 GRM1 NTRK1 SS18-SSX1 BRAF-FAM118B EWSR1-CREB1 HEY1-NCOA2 NTRK2 SS18-SSX2 BRAF-FRX1 EWSR1-CREB3L1 IGH-BCL2 NTRK3 SS18-SSX2 BRAF-GNA11 EWSR1-DDIT3 IGH-CCND1 NTRK3-ETV SS18-SSX6 BRAF-KIAA1549 EWSR1-DDIT4 IGH-CCND1 NUP98-HOXA13 TAF2N-NR4A3 BRAF-MACF1 EWSR1-E1AF IGH-CCND3 NUTM1-BRD2 TCF12-NR4A3 C11orf95-RELA EWSR1-ERG IGH-FGFR3 NUTM1-BRD3 TCF3-HLF CBFB-MYH11 EWSR1-ERG IGH-MAF NUTM1-BRD4 TCF3-PBX1 CDH11 EWSR1-ETV1 IGH-MAFB NUTM1-CIC TFE3-ASPL CDH11-USP6 EWSR1-ETV1 IGH-MYC NUTM1-YWHAE TFE3-ASPSCR1 CIC-DUX EWSR1-ETV4 IGK-MYC NUTM2B-YWHAE TFE3-MITF CIC-DUX4 EWSR1-FEV IGL-MYC NUTM2E-YWHAE TFE3-PRCC CIC-DUX4L1 EWSR1-FEV IRF4 PAX3-AFX TFEB-MALAT1 Gene fusion partners

Mertens, F., Johansson, B., Fioretos, T. et al. The emerging complexity of gene fusions in cancer. Nat Rev Cancer 15, 371–381 (2015). https://doi.org/10.1038/nrc3947 Mertens, F., Johansson, B., Fioretos, T. et al. The emerging complexity of gene fusions in cancer. Nat Rev Cancer 15, 371–381 (2015). https://doi.org/10.1038/nrc3947 Gene Fusion detection on DNA panels Referral: Atypical mesothelial proliferation Procedure: Biopsy Molecular findings Source: Primary STRN-ALK fusion Tumour: 85% (STRN exon 3 – ALK exon 20)

Pool: 898 Mean depth: 404

Breakpoints identified by MANTA on HPC Fusions by RNAseq

IlluminaTrusight RNA Pan Cancer 1500 targets Run in parallel with targeted DNA panels

PPM1G-ALK LMNA-NTRK1 Sequencing Workflow: RNA

Histopath. Library Prep Capture Analyse Sequence RNA (>20ng) Automatic 1st Hyb Demultiplex Sample taken Fragment FF 4.5 hrs samples DNA Cleanup Mapping with STAR aligner FF/FFPE 3 hrs 4 hrs

cDNA 2nd Hyb Illumina synthesis QC Picard Slide cutting Basespace

DNA Cleanup Assessment A-tailing Fusion calling H&E Manta Post Hyb-PCR Tissue Adapter 6 hrs (x11 cycles) Manual Check Isolation ligation (DI) 1 4 hrs RNA DNA Cleanup Extraction DNA Cleanup Manual Check 2 RNA QC Pre-hyb PCR Quantification and pooling (x15 cycles) Molecular Tumour Board DNA Cleanup Sequencing

2x75bp 3 Million Quantitate Report reads/sample

Preparation DAY 1-2 DAY 3-4 DAY 4 Fusions by long read technology: Oxford Nanopore CRISPR-CAS9 targeting

Oxford Nanopore Designing guide RNA for CRISPR-CAS9 targeting

PML exon X 5 – 15kb RARA exon 3 Genomic DNA chr15 chr17

Bound and protected

Ligate and Sequence Cas9

RARA exon3 PAM 5’ 3’ + strand GGGGCGTAGATGTTCGGAACGAA gRNA

3’ GATGGGGCGTAGATGTTCGGAACGAAACAGACAGTCCTGTTCAGGAGTCC 5’ - strand

Cut CRISPR- guide RNA design

Fusion site somewhere here

On-Target Off-Target SNP Design ID Gene /exon Position Strand Sequence PAM Score Score dbSNP ID Location GRCh38 No SNP CD.Cas9.BHZB9233.AC KMT2Ax5 221 + TAAAGAAAGGACGTCGATCG AGG 60 93 found N/A chr11:118478069-118478088 No SNP CD.Cas9.BHZB9233.AD KMT2Ax5 189 + CACTAGAAACAAGGCACCCC AGG 71 60 found N/A chr11:118478037-118478056 No SNP CD.Cas9.BHZB9233.AG KMT2Ax5 49 + TTGGAGTACCAATTAAAACC AGG 61 53 found N/A chr11:118477897-118477916 No SNP Hs.Cas9.RARA.1.AC RARAx3 113 - AAGCAAGGCTTGTAGATGCG GGG 52 71 found N/A chr17:40348403-40348384 No SNP Hs.Cas9.RARA.1.BA RARAx3 115 - CAAAGCAAGGCTTGTAGATG CGG 74 36 found N/A chr17:40348405-40348386 No SNP Hs.Cas9.RARA.1.AQ RARAx3 153 - GACCCCATAGTGGTAGCCTG AGG 52 58 found N/A chr17:40348443-40348424 No SNP CD.Cas9.VFQJ1967.AB ALKx20 287 - TGGCCTGTGTAGTGCTTCAA GGG 62 57 found N/A chr2:29223593-29223574 No SNP CD.Cas9.VFQJ1967.AH ALKx20 234 - ACCCACCTGCAGTGTACCGC CGG 41 84 found N/A chr2:29223540-29223521 No SNP CD.Cas9.VFQJ1967.AI ALKx20 282 - TGTGTAGTGCTTCAAGGGCC AGG 62 34 found N/A chr2:29223588-29223569 CRISPR-Cas9 enrichment experiments Nadine Holmes

Set 1: Proof of principle CRISPR Targets – KMT2A, RARA, ALK 3-5ug P/C or Qiagen extracted NA12878 genomic DNA ; sheared 20X with 26G needle > 500ng loaded on MinION or Flongle CRISPR-Cas9 enrichment experiments

Set 2: Cell lines, 2 cuts CRISPR Targets – KMT2A-AF9, PML-RARA, NPM1-ALK > 3 ug P/C extracted NB4 genomic DNA ; sheared 20X with 26G needle > 1000ng loaded on MinION on GridION mk1

: Multiplexing >Barcoded (Nanopore), Salted out or P/C cleaned up AMPure XP

Approx mount Amount added to Library Conc. in 24 ul barcoded Number Sample Extraction Shearing AMPure ng/ul (ng) Barcode pool 1 NB4 SaltOut 26G/20X 0.5X 41.2 988.8 NB01 400 ng 2 NB4 Ph:Chl 26G/30X 0.5X 137 3288 3 K299 Ph:Chl 26G/30X 0.5X 18.1 434.4 NB02 200 ng 4 K299 SaltOut 26G/20X 0.5X 68.4 1641.6 NB03 800 ng 5 Mac-1 SaltOut 26G/20X 0.5X 118 2832 NB04 800 ng 6 Mac-1 Ph:Chl 26G/30X 0.5X 56.4 1353.6 NB05 800 ng 7 Mac-2A Ph:Chl 26G/30X 0.5X 14.5 348 NB06 100 ng

K299 NMP-ALK +ve MAC-1 Control Cell lines NB4 PML-RARA +ve MAC2A Control Lewis Gallagher

CRISPR-Cas9 enrichment experiments

Set 3: Clinical Samples, 2 cuts PML-RARA CRISPR Targets – PML-RARA 1ug Standard NHS SOP (Qiagen) extracted patient genomic DNA 6 patients plus control loaded on MinION on GridION mk1

Amount of Sample Input Post barcoding Sample name Barcode each added Number amount QC (ng/ul) (ng)

1 19/06901 NB07 ~ 1ug 24 168 2 19/04983 NB08 ~ 1ug 40.6 284 3 18/12130 NB09 ~ 1ug 26.6 186 4 18/08651 NB10 ~ 1ug 32.2 225 5 17/01162 NB11 ~ 1ug 29.6 207 6 16/04482 NB12 ~ 1ug 45.6 319 19/06901 NB07 PML exon 6: RARA exon 3

Actual breakpoints 1906901_NB0 7 chr15 + 74033832 chr17 40348170 8 20 chr15 - 74033832 chr17 40348170 8 20 chr15 + 74033848 chr17 40343975 1 20 chr15 - 74033826 chr17 40348171 1 20 chr15 - 74033830 chr17 40348170 1 20 chr15 - 74033848 chr17 40343975 1 20

PML CRISPR ex3 RARACRISPR ex3 1904983_NB08 PML exon 6: RARA exon 3

Actual breakpoints PML exon 6 (exonic): RARA exon 3

chr15 + 74033358 chr17 40340742 1 3 chr15 + 74033848 chr17 40343975 1 3 chr15 - 74033848 chr17 40343975 1 3

PML CRISPR ex3 RARACRISPR ex3

1904983_NB08 chr15 + 74033358 chr17 40340742 1 3 chr15 + 74033848 chr17 40343975 1 3 chr15 - 74033848 chr17 40343975 1 3 1812130_NB09 PML exon 3: RARA exon 3

PML CRISPR ex3 RARACRISPR ex3

1812130_NB09 chr15 + 74024546 chr17 40332946 21 34 chr15 - 74024546 chr17 40332946 6 34 chr15 + 74024534 chr17 40332959 1 34 chr15 + 74024545 chr17 40332946 1 34 chr15 + 74024545 chr17 40332949 1 34 chr15 + 74024545 chr17 40332973 1 34 chr15 - 74024521 chr17 40332967 1 34 1808651_NB10 PML exon 6: RARA exon 3

PML CRISPR ex3 RARACRISPR ex3

1808651_NB10 chr15 - 74033848 chr17 40343975 18 49 chr15 + 74033848 chr17 40343975 15 49 1701162_NB11 PML exon 3: RARA exon 3

PML CRISPR ex3 RARACRISPR ex3

1701162_NB11 chr15 + 74024703 chr17 40338759 2 7 chr15 - 74024542 chr17 40332946 1 7 chr15 - 74024546 chr17 40332946 1 7 chr15 - 74024703 chr17 40338759 1 7 chr15 - 74024703 chr17 40338766 1 7 chr15 - 74033821 chr17 40348170 1 7 1604482_NB12 PML exon 6: RARA exon 3

PML CRISPR ex3 RARACRISPR ex3

1604482_NB12 chr15 + 74034380 chr17 40334835 2 8 chr15 + 74033831 chr17 40348170 1 8 chr15 + 74033848 chr17 40343975 1 8 chr15 + 74034375 chr17 40334842 1 8 chr15 - 74033833 chr17 40348170 1 8 chr15 - 74034375 chr17 40334835 1 8 chr15 - 74034380 chr17 40334839 1 8 Targeting Multiplex Low-input (10ng) WGA What’s next?

• Validation (Sensitivity, Specificity, Reproducibility, Replicability) • Criteria for calling a negative? • Enrichment, reduction of off target • Speed up, lower cost (read-until?) • Improve assay – optimize multiplex targets • Flongle? Other Nanopore platforms? • Combine with other SV assays (e.g. ITD, InDel) • -GridION onsite…..