Gene Targeting of the HBB Locus by Crispr/Cas9 to Investigate Repair Pathway Choice in Response to Different Types of DNA Lesions C

Gene targeting of the HBB locus by Crispr/Cas9 to investigate repair pathway choice in response to different types of DNA lesions C. Cotta-Ramusino, T. Phadke, M. Maeder, D. Bumcrot Editas Medicine, Inc., Cambridge MA 02142

HBB" HBD" HBG1" HBG2" Chr.11 Nickase N863A_Cas9 leads to higher 8 β" δ" Aγ" Gγ" ε" HBB ~6kb" levels of insertions 15 Introduction D- C- B- A- A+ B+ C+ Adult Fetal Embryonic LCR HBD 40bp 50bp Sickle Mutation Frequency of Insertions 120bp 47 bp 260bp 130bp 50% Cut Cas9 WT 8 gRNA NGG region The CRISPR/Cas9 system has recently been 40% 5’ propelled to the forefront of the genome editing field Sickle Mutation 30% Lenth of the Gene Conversion as a fast and reliable method for introducing 70% N863A 8 gRNA NGG 20% 60% targeted DNA double-strand breaks into the 3’ 50% 40% genome. Derived from a bacterial adaptive immune 3’ 10% GGN 15 gRNA 30% 20% system, this technology uses short guide RNAs 0% 10% Frequency Cas9 WT_8 N863_8/15 D10A_8/15 0% (gRNAs) to direct the cleavage activity of the Cas9 A+/ D10A 8 gRNA NGG D- C- B- B+ C+ 5’ A- protein in a site-specific manner. Inactivating point N863A Cas9 insertions are mostly duplication of the 3’ 5’ D10A_8/15 14% 12% 14% 60% 0.5% 0% mutations engineered into either the HNH or RuvC GGN 15 gRNA protruding arm catalytic domains enable conversion of Cas9 from a nuclease to a single-strand nickase. Fig.2 Fig.5 Fig.8 Here we investigate the use of this powerful genome Schematic of the HBB locus indicating the position of Frequency of insertion observed. Results are Frequency with which different lengths of HBD editing technology to target the human HBB gene in the different gRNAs used in the study. compiled from 3 independent experiments for each sequence are incorporated into HBB. the region of the sickle cell anemia-causing condition tested. mutation. Sickle Cell Anemia is a recessive disorder caused by a single point mutation in the human beta Different Cas9 variants cut with a Examples of the Insertion after nicking D10A Cas9 induces higher rate of HDR with globin gene. comparable aggregate efficiency with N863A lower strand ssDNA as donor template We demonstrate the use of the CRISPR/Cas9 HDR system to target the human HBB gene and examine HBB 30% 25% how the nature of the targeted break affects the 20% All editing events 15% frequency of different DNA repair outcomes. 10%

100% Insertion Example Utilizing the wild type Cas9 nuclease, as well as two Sequence 5% 0% 80% different Cas9 nickases we are able to introduce Lower Lower Upper Lower Upper strand strand strand strand strand blunt double-strand breaks, single-strand nicks, and 60% WT N863A D10A dual-nicks in which the nicks are placed on opposite 40% 8 strands and leave either 3’ or 5’ overhangs of HBB 20% 15 47bp varying lengths. Using either single-strand 60bp 60bp 0% DONOR oligonucleotide (ssODN) or plasmid DNA donors, we Cas9 WT_8 N863_8/15 D10A_8/15 60bp: 8 mismatch characterize several different DNA repair outcomes including indel mutations resulting from non- homologous end-joining, homology-dependent Fig.3 Fig.6 Fig.9 Total editing observed with three Cas9 variants. Example of common Sanger reads observed in Frequency of HDR in response to different donors. repair (HDR) using the donor as a template, and Results are compiled from at least 3 independent U2OS cells electroporated with Cas9 N863A and Schematic illustrating the donor template is shown HDR using the closely related HBD gene as an experiments for each condition. gRNA 8/15. The HBB reference is indicated on the on the bottom. endogenous template. The frequency with which we top. observe these various repair outcomes under Methods: different conditions offer insight into the mechanisms WT _Cas9 leads to Nickase D10A_Cas9 leads to higher U20S cells were electroporated with gRNA and of DNA repair and how it is impacted by the nature higher levels of deletions levels of Gene Conversion from HBD Cas Wt or mutant plasmid. Cells were collected 6 of the DNA break. The data also suggests a days after electroporation. gDNA was extracted, potential therapeutic approach in which correction of Frequency of Deletion Gene Conversion PCR amplification of HBB locus was performed 70% 35% the sickle-cell mutation is efficiently mediated 30% and sub cloned into a Topo Blunt Vector. For each 60% through HDR with either a donor template or with 25% condition in each experiment 96 colonies were 50% 20% the HBD gene. 15% sequenced by Sanger sequencing. 40% 10% 30% 5% Conclusion: Fig 1: Schematic of Cas9 variants used in this study 0% 20% Cas9 WT_8 N863_8/15 D10A_8/15 • Cas9 wt, N863A and D10A cut with a 10% 8 HBB 15 comparable efficiency 0% 6Kb Cas9 WT_8 N863_8/15 D10A_8/15 D- C- B- A- A+ B+ C+ HBD • Cas9 N863A Nickase induces a higher rate of Cut • With Cas9 WT deletions are mostly less then 5 bp region insertions 15 8 • With Cas9 Nickase deletions are mostly around 40-50 bp HBB HBD • Cas9 D10A Nickase induces modification of the Cluster of sequencing difference HBB locus using the HBD gene as a donor Fig.4 Fig.7 template Frequency of deletion observed with three Cas9 Frequency of gene conversion. A representation of - Predicted to repair the sickle mutation variants. Results are compiled from 3 independent at least 3 independent experiments for each • Cas9 D10A Nickase achieves a higher rate of experiments for each condition. condition is shown. Schematic representing the HDR genomic organization and the region of similarity between HBB and HBD is shown on the bottom.