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 C 0 .5 C 0 .5 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 M 4 0 M n M M 4 0 4 0 M n n 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 C 0 .5 a 8 0 % e 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 C 0 .5 0 .5 i 7 0 % t 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.