Conflict of Interest Declaration. Targeted Genome Editing. Applications of Genome Editing Technology 5/4/2016

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1 Conflict of Interest Declaration JKJ has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals JKJ is a consultant for Horizon Discovery JKJ s interests were reviewed and are managed by Massachusetts eneral Hospital and Partners HealthCare in accordance with their conflict of interest policies Targeted enome Editing capability to introduce targeted alterations into NY specific gene sequence with high efficiencies in NY living cell or organism pplications of enome Editing Technology For research applications: generating customized cell lines for disease modeling developing animal models with mutated disease genes For therapeutics: knocking out, knocking in, or correcting genes could be broadly useful for treating genetic diseases 1

2 Targeted DN Breaks Enable Targeted enome Editing enome Editing Nucleases Zinc Finger Nucleases (ZFNs) TLE Nucleases (TLENs) Meganucleases CRISPR RN uided Nucleases Challenge: how does one make the required targeted double stranded break? RN uided CRISPR Cas9 Nucleases further simplifies targeting of nucleases uses simple base complementarity C C U U C C worked in vitro but would it work in vivo and in cells? grn U U U C U C Cas9 U U U C U C U U U U C C C C U C U C U U U C U C U U U U CCTCCTCCCTCCCTT N N N N N N 3 -NNNNNNNNNNN NNNNNNNNNNN-5 5 -NNNNNNNNNNN NNNNNNNNNNN-3 N N N N CCCTC PM Target site Jinek et al., Science 2012 Pennisi, Science 2013 Sander & Joung, Nat Biotechnol

3 CRISPR Cas9 Specificity Define Improve Change So targeted genome editing is now simple with CRISPR But what about off target effects? Important issue to understand for both research and therapeutic applications In particular for therapeutics, the ideal method for nuclease offtarget identification would be: (1) genome wide, (2) unbiased, and (3) highly sensitive Finding Off Target Mutations Using In Silico Programs + Focused Sequencing Fu et al., Nat Biotechnol

4 Finding Off Target Mutations Using In Silico Programs + Focused Sequencing enomewide Unbiased Identification of DSBs Enabled by sequencing (UIDE seq) 1. Tag DSBs with dsodns (likely by NHEJ) Mutt and Jeff Comic Strip, Florence Morning News, 6/3/1942 from: light/ 2. Unbiased amplification of one side of the tag 3. Sequence and map amplified fragments Tsai et al., Nat Biotechnol Testing UIDE seq With Ten grns Target site Cells Target Site (5' --> 3') name EM1 U2OS TCCCN VEF site1 U2OS TTTTCTCCN VEF site2 U2OS CCCCCTCCCCCCCCTCN VEF site3 U2OS TTTTTCTN RNF2 U2OS TCTCTTTCTTCCTN FNCF U2OS TCCCTTCTCCCCN HEK293 site CCCCTCCTN HEK293 site CCCTCTCN HEK293 site CCCCTCCTN HEK293 site CCTCCTTN used four grns for which we and others had previously identified some off target mutation sites (found by simple mismatch counting) used two different human cell lines enome wide Off Target Profiles of CRISPR Cas9 Tsai et al., Nat Biotechnol

5 Take Home Messages UIDE seq is genome wide, unbiased, and highly sensitive sensitivity for mutations as low as 0.1% frequency UIDE seq potentially important for assessing therapeutic Cas9 nucleases also provides a method to measure and quantify alterations and improvements to Cas9 specificity however, I do NOT recommend routine use of UIDE seq (or other methods) for most research experiments use controls instead! Tsai et al., Nat Biotechnol CRISPR Cas9 Specificity Define Previously Described Methods for Improving CRISPR Cas9 Specificity Mali et al., Nat Biotechnol uilinger et al., Nat Biotechnol Improve Ran et al., Cell 2013 Tsai et al., Nat Biotechnol Kleinstiver et al., Nature 2015 Change Fu et al., Nat Biotechnol Slaymaker et al., Science 2016 ll previously described methods leave residual detectable off target mutations and/or have not been assessed using a highly sensitive genome wide method 5

6 Mutating Non Specific DN Contacts Mediated by SpCas9 ctivities of lanine Substituted SpCas9 Variants at On Target Sites ctivities of lanine Substituted SpCas9 Variants at Mismatched Sites ctivities of lanine Substituted SpCas9 Variants at Mismatched Sites 6

7 On Target ctivities of SpCas9 HF1 at Endogenous Human ene Sites enome wide Off Target Profiles of SpCas9 HF1 enome wide Off Target Profiles of SpCas9 HF1 Confirmatory Targeted Deep Sequencing of SpCas9 HF1 Off Target Sites 7

8 Confirmatory Targeted Deep Sequencing of SpCas9 HF1 Off Target Sites SpCas HF1 can be used instead of wild type SpCas9 to minimize off target effects for most applications further specificity improvements will likely require development of more sensitive methods for detecting genome wide off target mutations CRISPR Cas9 Specificity Define PM Requirement Limits SpCas9 Targeting Range Improve Change 8

9 PM Requirement Limits SpCas9 Targeting Range Evolving CRISPR-Cas9 Variants Using Rational Design and Bacterial-based Selection Nearest wild-type SpCas9 20-mer site is 53 nucleotides away Precise DSB targeting capability is important for: Homologous recombination efficiency (DSB within 10 bp of desired change) Targeting of small genetic elements such as: microrns, splice sites, transcription-factor binding sites, short ORFs/micro peptides, etc. Exploiting PM recognition to achieve single base allele discrimination nders et al., Nature 2014 dditional Rational Design & Testing Kleinstiver et al., Nature 2015 Evolved SpCas9 Variants Enable Editing of Endogenous Human ene Sites PM Requirement Limits SpCas9 Targeting Range Kleinstiver et al., Nature

10 Evolved Variants Expand SpCas9 Targeting Range enome-wide Specificity of Engineered SpCas9 Variants can now precisely target at site of interest but what about the genome wide specificities of these evolved variants? Kleinstiver et al., Nature 2015 Using Evolved SpCas9 Variants Engineered variants increase the targeting range of SpCas9 and SaCas9 CasBLSTR: web based tool for finding target sites with N and NC PMs (or PMs of other Cas9 orthologues or custom PMs) ll Reagents vailable at ddgene cas 10

11 Future Challenges for enome Editing CRISPR/Cas Components Efficiency and Specificity Cellular Determinants of ctivity Effective Delivery pproaches Jose James Ibrahim Jackson Maggie Esther Becca Stacy Moira Not pictured:, and Stephanie Collaborators: Martin ryee, John Iafrate, Randy Peterson, Joanna Yeh (MH) Slide courtesy of Sandra lucksmann, Editas Medicine Mutating Non Specific DN Contacts Mediated by SpCas9 Comparisons of UIDE seq with Computational Prediction Software CRISPR Design Tool E CRISP Tsai et al., Nat Biotechnol

12 Methods for enome wide Off Target Determination IDLV Capture BLESS UIDE seq HTTS Digenome seq adapted from abriel et al., Nat Biotechnol. 2015, Kim et al., Nat Methods 2015 & Ran et al., Cell 2015 Evolving Staphylococcus aureus Cas9 (SaCas9) from Nishimasu et al., Nature 2015 SaCas9 is more than 300 aa smaller than SpCas9 advantages for packaging into viral vectors such as V SaCas9 s longer PM (NNRRT) restricts targeting range 1 in 32 bps of random DN vs. 1 in 8 bps for SpCas9 Evolving Staphylococcus aureus Cas9 (SaCas9) oal: Increase Targeting Range by Relaxing SaCas9 PM specificity NNRRT (1 in 32 bps) Predicting SaCas9 s PM interacting domain: ctivity & Specificity of Evolved KKH SaCas9 in Human Cells On target activities in human cells: NNNRRT (NNRRT NNRRT NNTRRT NNCRRT) (1 in 8 bps) Mutagenesis and selection: Specificities in human cells as judged by UIDE seq: KKH SaCas9 variant Kleinstiver et al., Nat Biotechnol., 2015 Kleinstiver et al., Nat Biotechnol.,