Amplicons, Heteroduplexes and Enzymes - Proper Processing Elevates Detection of CRISPR Gene Editing Events

Transcription

1 Amplicons, Heteroduplexes and Enzymes - Proper Processing Elevates Detection of CRISPR Gene Editing Events Steve Siembieda, MS MBA VP Commercialization ABRF Conference February 2015

2 What Is CRISPR? Clustered Regularly Interspaced Short Palindromic Repeats An adaptive defense mechanism in bacteria First described by Yoshizumi Ishino in 1987 Cas9 nuclease is derived from Streptococcus pyogenes Cleavage, interference, activation

3 Simplicity is Revolutionizing Gene Editing Requires 2 components 1. Synthetic RNA molecules called sgrna (single guide RNA) complementary to a target gene sequence -The only constraint is the need for a terminal NGG. Can be multiplexed 2. CRISPR-associated gene 9 (cas9) nuclease -Same for all sites targeted -Can be expressed from vector

4 Experimental Design Challenge Cas9 considerations Cas9 protein Cas9 mrna Stably integrated cell line Plasmid expression Lentivirus expression Nickase, double nickase or fully active Cas9 New Cas analogues Guide RNA considerations Single guide RNA One RNA construct (guide + tracr) Two guide RNA Guide separate from tracr Synthetic RNA oligos Expressed in cells (plasmid, gblock, viral) Transcribed In vitro

5 Experimental Design Challenge Cas9 considerations Cas9 protein Cas9 mrna Stably integrated cell line Plasmid expression Lentivirus expression Nickase, double nickase or fully active Cas9 New Cas analogues Guide Donor RNA DNA considerations considerations Single guide RNA Single strand oligo One RNA construct (guide (ssodn) + tracr) Two guide RNA Double Guide strand separate fragment from tracr Length Synthetic Creation RNA oligos Expressed Delivery/Expression in cells (plasmid, gblock, viral) Transcribed In vitro

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7 Small Guide RNA target design 95% of sites 90% 93% 86% 75% 97%

8 The Repair Mechanism NHEJ NHEJ (Non-Homologous End Joining) -Error prone process -Random mutation, predominately single point indels -Typically a high frequency event HDR (Homologous Directed Repair) -Use of donor DNA to create specific mutational changes at the cleavage site such as 1) Correcting known mutations 2) Adding foreign DNA 3) Excising defined fragment -Low frequency event, <5% 8

9 Mutation Frequency Determination Is an important step Understand what/if worked How to move forward decision time Different ways to detect mutation Heteroduplex cleavage PCR amplification Sequencing

10 Heteroduplex Cleavage A simple method to use Multiple things to consider Adding in wild type DNA is important A standardized protocol is needed

11 Optimization Considerations Effects on cleavage efficiency Pools vs individual cells Heteroduplexing protocol DNA to enzyme ratio Enzyme used Cleavage time Amplicon length Equipment used Zygosity determination

12 Experimental Design Plasmid based system synthetic gene Perfect ±1, ± 2 and ± 10 indels All 10 point mutations (SNP)

13 Experimental Protocol Amplify a fragment from the WT and mutated plasmids Quantify amplicon Combine at ratios to 10ng/µL final conc Heteroduplex Cleave Separate on Fragment Analyzer Calculate cleavage rate

14 Heteroduplex Protocols 600bp amplicon at 50:50 mixture T7 endonuclease RAMP protocol (~20 minutes) 95 C for 5min 95 C-85 C at -2 C/sec 85 C-25 C at -0.1 C/sec STEP protocol (~1 hour) 99 C for 5min 2-step cycle protocol -Step 1-70 C for 20sec, reduce by 0.6 C each cycle -Step C for 20sec, reduce by 0.6 C each cycle -Repeat 2 step cycle 70

15 Heteroduplex Protocols 600bp amplicon at 50:50 mixture T7 endonuclease RAMP protocol (~20 minutes) 95 C for 5min 95 C-85 C at -2 C/sec 85 C-25 C at -0.1 C/sec STEP protocol (~1 hour) 99 C for 5min 2-step cycle protocol -Step 1-70 C for 20sec, reduce by 0.6 C each cycle -Step C for 20sec, reduce by 0.6 C each cycle -Repeat 2 step cycle 70

16 Enzyme Comparison 600bp amplicon at 50:50 mixture Ramp protocol T7 Endonuclease 4µl DNA at 10ng/µl -40ng total DNA -1µl T7 Surveyor 8µl DNA at 50ng/µl -400ng total DNA -1µl Enhancer -1.6 µl 0.15M MgCl2-1µl Surveyor

17 Enzyme Comparison 600bp amplicon at 50:50 mixture Ramp protocol T7 Endonuclease 4µl DNA at 10ng/µl -40ng total DNA -1µl T7 Surveyor 8µl DNA at 50ng/µl -400ng total DNA -1µl Enhancer -1.6 µl 0.15M MgCl2-1µl Surveyor

18 Amplicon Size Comparison 200 bp-1,000 bp Ramp protocol T7 endonuclease 50:50 mixture 75:25 mixture

19 Amplicon Size Comparison 200 bp-1,000 bp Ramp protocol T7 endonuclease 50:50 mixture 75:25 mixture

20 Cleavage Rate & Mutation Frequency Cleavage rate mutation frequency %cleavage = average molarity (frag1 + frag2) average molarity(frag1 + frag2) + molarity (uncut) Mutation frequency calculation Individual % mutated = 50-5* % cleaved Pooled % mutated = 1-1 % cleaved

21 Cleavage Rate & Mutation Frequency Mutated Cell WT Cell 10% mutated 90% WT Amplify cells individually Heteroduplex Post amplification quantification Theoretical % Cleavage Theoretical % Mutated 0% 0% 18% 10% 32% 20% 42% 30% 48% 40% 50% 50%

22 Theoretical vs Actual Clevage

23 Cleavage Rate & Mutation Frequency

24 Cleavage Rate & Mutation Frequency

25 PROSize Plug-in Zygosity Di

26 Mutation Detection Kits DNF CP Mutation Discovery Gel dsdna reagent kit Analysis range bp Enzyme Kit - In development For automated digestion of heteroduplex fragments Optimization completed Available soon 26

27 Fragment Analyzer Plays Unique an design important features role Premier improve automation laboratory workflow instrument Only Ideal automated for shared use platform labs with CRISPR Three 96 detection well plates kits gives No maximum issue with flexibility digestion buffers High Achieve detection results sensitivity confidently CRISPR-specific Solution for customers software tools

28 Fragment Analyzer Plays an important role Premier automation instrument Only automated platform with CRISPR detection kits No issue with digestion buffers High detection sensitivity CRISPR-specific software tools

29 CRISPR Support Booth 509 Crisprupdate.com New CRISPR poster

30 CRISPR Support Booth 509 Crisprupdate.com New CRISPR poster