An in Vitro Method for Benchmarking of CRISPR-Associated Endonucleases

An in vitro method for benchmarking of CRISPR-associated endonucleases

Eric Tillotson1, Gregory Gotta1, Katherine Loveluck1, John Zuris1, Barrett Steinberg1, Tongyao Wang1, Ramya Viswanathan1, Elise Keston-Smith1, Joost Mostert1, Luis Barrera1, Christopher Wilson1, Vic Myer1, Hari Jayaram1

1Editas Medicine, 11 Hurley Street, Cambridge, MA. 02141

Introduction In vitro Benchmarking of CRISPR-associated Nucleases Direct delivery of Cas9 or Cpf1 ribonucleoprotein (RNP) complexes to a cells by electroporation is emerging as an important delivery mode for ex D N M T 1 _ 0 1 D N M T 1 _ 2 6 D N M T 1 _ 1 6 2 P D 1 _ 0 2 P D 1 _ 0 8 1 .0 1 .0 1 .0 1 .0 1 .0 vivo gene editing therapies. Understanding the dose/efficacy relationship S p S p S p S p S p

d S a u d S a u d S a u

d S a u d S a u e e e

of the delivered RNP is a key step in optimizing a therapeutic. An e e v v v v v a a H iF i a H iF i H iF i a H iF i a H iF i e e e l l e e l essential requirement in accurately establishing this relationship and l l

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C 0 .5

C 0 .5 e c a s e c a s C 0 .5 e c a s e c a s e c a s

n n n n n o o o i i o o i

determining RNP quality is measuring the in vitro activity of a given RNP i i t a s w t t A s R R t A s R V R

t a s w t t A s R R c c c c c a a a a a r r r r L b L b r complex in a standardized manner. Here we describe an in vitro assay F F F F F F n F n for measuring the specific activity of CRISPR-associated nucleases on 0 .0 0 .0 0 .0 0 .0 0 .0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 synthetic DNA substrates. We then apply this assay to benchmark the R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) activity of several naturally occurring and engineered CRISPR P D 1 _ 0 2 nucleases. This standardized in vitro benchmarking provides critical b D N M T 1 _ 1 6 2 D N M T 1 _ 0 1 D N M T 1 _ 2 6 P D 1 _ 0 8 6 0 8 0 6 0 input to help create the best RNP based therapy. 6 0 6 0

5 0 5 0 5 0 5 0 Specific Activity Method 6 0

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n 4 0 n 4 0 The specific activity assay substrate (a) is a 200 bp synthetic DNA 3 0 3 0 amplicon comprised of genomic DNA target sequences and constant 5’ 3 0 3 0 2 0 2 0 2 0 and 3’ primer handles. Selected target sites have a 3’ NGGRRT PAM 2 0 2 0 9 9 1 9 R 9 9 1 s s F s R 9 9 1 9 1 1 1 9 R a a a - 9 9 1 9 R f f f 9 9 1 1 1 1 s s F s V H s s F s s s F s p 9 f f f a a a C C - C f1 R a a a p p s s F s p H R p u 9 a a H a - -H C C C a a a p p C C - C - s p p C C - 1 with one of the following 5’ PAMs 20 bp upstream: TTTV, TTN, NYCV, or C C C s H C p u 9 1 S a S 9 C f p u 9 p b n C C - C C C s p f S a C p u p p s A L F p u 9 s b n S a S p e s a s S a S s p A S a C S a S C S a e S a S L F C e C p A S e C S a C s e p s S A p C A p TATV, allowing us to compare activities of multiple nucleases using the S p S S S same synthetic substrate. EC50 values are generated by a dose- response of RNP over 15 nM substrate for 1 hour at 37º C in 30 mM a) Specific activity benchmarking was performed on the following CRISPR nucleases: SpCas9, SauCas9, High-fidelity (SpCas9-HF1), enhanced specificity (eSpCas9), AsCpf1, LbCpf1, FnCpf1, AsCpf1-RVR, and AsCpf1 RR. N = 2 b) 95% CI EC50 values indicate that the variance in activity between enzymes is similar and largely target specific. NGG PAM was used for SpCas9, SpCas9-HF1, and eCas9. Hepes pH 7.5, 150 mM NaCl, and 2 mM MgCl2.. RNP is added to DNA NGGRRT for SauCas9. TTTV for AsCpf1, LbCpf1, and FnCpf1. TATV for AsCpf1 RVR. NYCV for AsCpf1 RR. substrate using a Biomek FXp liquid handler. Nuclease activity is quenched by addition of Proteinase K. Fraction of cleaved substrate is measured using the Fragment Analyzer automated CE system (AATI). Analysis is performed with GraphPad Prism using four parameter dose-response. Assay Characterization and Sensitivity Off-target Analysis of Engineered Variants a 20mer Target 5’ 3’ Cpf1 variant PAM Sp/Sau Cas9 PAM a) Assay sensitivity was a a i ii determined by doping in of i. O n T a r g e t O ffT a rg e t 1

All RNPs were formed at 2:1 in excess of gRNA in 30 mM Hepes pH 7.5, D e a d g R N A D o p in g d S p C a s 9 D o p in g 1 .0 1 .0 dead (orthogonal) gRNA and S p C a s 9 S p C a s 9 150 mM NaCl, and 2 mM MgCl . Guides were supplied by IDT as 2-part 1 .0

2 1 0 0 % d e S p C a s 9 1 .0 1 0 0 % w tC a s 9 ii. dSpCas9 b) 95% d e S p C a s 9 e e v v a alt-R for SpCas9, SauCas9, SpCas9-HF1, and eSpCas9. IDT single d 9 0 % S p C a s 9 -H F 1 a S p C a s 9 -H F 1 d 9 0 % e e l confidence interval values e e v l v a 8 0 % C 0 .5

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guide alt-R were used for AsCpf1, LbCpf1, FnCpf1, AsCpf1-RVR, and l n e n l C 7 0 % indicate EC50 values are o

i o

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n t c n c AsCpf1 RR. Digital scanning fluorimetry (DSF) was performed on RNPs o i a o

t 6 0 % a i r

t 6 0 % significantly different at 50% r c F c a F r 5 0 % a to ensure RNP complexation. r F 5 0 % F i. dead gRNA and ii. 0 .0 2 5 % 0 .0 0 .0 2 5 % 0 .0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 0 % dSpCas9 c) Decrease in 1 1 0 1 0 0 1 0 0 0 1 0 0 % d C a s 9 R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) Conclusions R N P T re a tm e n t (n M ) R N P T re a tm e n t (n M ) Amax suggests dSpCas9 In vitro matched site benchmarking offers activity comparisons between binds to substrate DNA O ffT a rg e t 2 O ffT a rg e t 3 inhibiting SpCas9 activity. 1 .0 1 .0 CRISPR-associated nucleases. Potency measurements on matched S p C a s 9 S p C a s 9

d e S p C a s 9

d e S p C a s 9 e substrates indicate no significant differences in activity across nucleases ii e v b i v c a S p C a s 9 -H F 1 a S p C a s 9 -H F 1 e e l tested. Finally, engineered variants High-fidelity Cas9 and enhanced d S p C a s 9 D o p in g A m a x l D e a d g R N A D o p in g E C 5 0 d S p C a s 9 D o p in g E C 5 0 C 0 .5

C 0 .5

n n o o 1 .2 i i specificity Cas9 display target discrimination in vitro. Benchmarking 2 0 0 6 0 t t c c a a r 1 .0 r F

these nucleases enables understanding the cellular dose-efficacy F 1 5 0 5 0 relationship and helps optimize an ex-vivo RNP therapeutic. 0 .8 0 .0 0 .0

x 1 1 0 1 0 0 1 0 0 0 1 1 0 1 0 0 1 0 0 0 a M M 0 .6 R N P T re a tm e n t (n M ) 4 0 m R N P T re a tm e n t (n M ) n 1 0 0 n A Disclosure: This presentation contains forward-looking statements within the meaning of the “safe harbor” provisions of The Private Securities Litigation Reform Act of 1995. All statements, other than statements of historical facts, 0 .4 contained in this presentation, including statements regarding the Company’s strategy, future operations, future financial position, future revenue, projected costs, prospects, plans, and objectives of management, are forward- looking statements. The words ‘‘anticipate,’’ ‘‘believe,’’ ‘‘continue,’’ ‘‘could,’’ ‘‘estimate,’’ ‘‘expect,’’ ‘‘intend,’’ ‘‘may,’’ ‘‘plan,’’ ‘‘potential,’’ ‘‘predict,’’ ‘‘project,’’ ‘‘target,’’ ‘‘should,’’ ‘‘would,’’ and similar expressions are 3 0 intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. Forward-looking statements in this presentation include statements regarding the Company’s goals of 5 0 0 .2 a) SpCas9, SpCas9-HF1, and eSpCas9 show comparable on-target activity. Rationally submitting of an IND for the LCA10 program in 2017, initiating an LCA10 natural history study in mid-2017, achieving preclinical proof-of-concept for additional programs and establishing alliances. The Company may not actually achieve the plans, intentions, or expectations disclosed in these forward-looking statements, and you should not place undue reliance on these forward-looking statements. Actual results or events could differ materially 2 0 0 .0 engineered SpCas9-HF1 and eSpCas9 have almost no activity against off-targets in from the plans, intentions and expectations disclosed in these forward-looking statements as a result of various factors, including: uncertainties inherent in the initiation and completion of preclinical studies and clinical trials and

e e e e e e clinical development of the Company’s product candidates; clinical trials; whether interim results from a clinical trial will be predictive of the final results of the trial or the results of future trials; expectations for regulatory % % % % e e e e e e e % % 0 0 0 0 % e v v v v v v v iv iv iv iv iv iv 0 0 8 7 6 5 5 v li li li li li li i l l l l l l vitro. approvals to conduct trials or to market products; availability of funding sufficient for the Company’s foreseeable and unforeseeable operating expenses and capital expenditure requirements; and other factors discussed in the 0 9 2 li l 1 % % % % % % % % % % % % % % 0 0 0 0 0 5 “Risk Factors” section of the Company’s most recent Quarterly Report on Form 10-Q, which is on file with the Securities and Exchange Commission, and in other filings that the Company may make with the Securities and 0 0 0 0 0 0 5 0 9 8 7 6 5 2 0 9 8 7 6 5 2 0 Exchange Commission in the future. In addition, the forward-looking statements included in this presentation represent the Company’s views as of the date of this presentation. The Company anticipates that subsequent events 1 1 and developments will cause its views to change. However, while the Company may elect to update these forward-looking statements at some point in the future, it specifically disclaims any obligation to do so. These forward- looking statements should not be relied upon as representing the Company’s views as of any date subsequent to the date of this presentation.