Alt-Ring the genome with CRISPR- Cas9 Genome editing done easily and fast CHIA Jin Ngee Regional Application Specialist 1
Integrated DNA Technologies The Custom Biology Company 2
IDT was founded by a scientist for scientists Founded in 1987 by Dr Joseph Walder >1000 employees in 6 locations 4 manufacturing sites Dedicated R&D facilities Active basic research Largest custom nucleotide manufacturer worldwide 3
IDT is the largest oligonucleotide manufacturer worldwide enabling new discoveries in biology and medicine >82,000 active customers >75,000 oligos synthesized per day >3,500 orders per day 300,000 web site visits per month 4
With our global facilities, the sun never sets on IDT Redwood City, CA, USA 743 m 2 Dedicated R&D site Development of genomics applications in NGS and qpcr Leuven, Belgium 2660 m 2 ISO 9001:2008 certified Europe, Africa, and Middle East production and support San Diego, CA, USA 1654 m 2 ISO 9001:2008 certified Production and next-day delivery for western United States Coralville, IA, USA (Headquarters) 14,300 m 2 Global infrastructure, production, and support offices ISO 9001:2008 certified ISO 13485:2003 certified and FDA-registered clean room: 310 m 2 Software development and IT: 2200 m 2 Product R&D: 1028 m 2 Skokie, IL, USA Administrative and financial office Singapore 804 m 2 bizsafe certified Asia-Pacific production and support 5
5 drivers of IDT s leadership in custom oligonucleotide manufacturing Vertical integration Broad nucleic acid portfolio Dedication to high quality Speed of delivery Unrivalled customer service Design and build proprietary DNA synthesizers In-house formulation of all key synthesis reagents Standard DNA oligos Modified oligos RNA High volume plate orders 100% QC of oligos by ESI mass spec Daily monitoring of synthesis quality and manufacturing equipment >90% of orders are shipped in <24 hours >3,500 packages shipped each day >165,000 customer support interactions annually Most widely used online oligo design and analysis tools Creation of proprietary manufacturing software Large scale Double-stranded DNA (gblocks fragments) Custom genes 6
High automation allows fast turnaround times of quality nucleic acid products 7
Our proprietary, automated synthesis platforms increase quality and speed, with less chance of human error 8
Have a bag of crispy CRISPR Clustered Regularly Interspaced Short Palindromic Repeat Adapted to edit genomes 9
Significance of CRISPR There are ~ 25,000 annotated genes in the human genome
Seen this film? Released 2 May 1998 Stars Ethan Hawke, Uma Thurman and Jude Law Vincent Freeman (Ethan Hawke) has always fantasized about traveling into outer space, but is grounded by his status as a genetically inferior "in-valid." He decides to fight his fate by purchasing the genes of Jerome Morrow (Jude Law), a laboratory-engineered "valid." He assumes Jerome's DNA identity and joins the Gattaca space program, where he falls in love with Irene (Uma Thurman).
Engineering a valid just like in Gattaca
More on engineering an in-valid to a genetic valid
Timeline of CRISPR-Cas Genome Engineering CRISPRs first described 1987 CRISPR-Cas identified as an immune system for bacteria 2007 CRISPR-Cas9 is RNA-guided DNA endonuclease (Jinek et al.) 2012 2005-2006 CRISPRs comprised of viral sequences 2010-2011 Characterization of type II CRISPR-Cas systems cutting DNA, identification of tracrrna 2013 Cas9-RNA used for genome editing in human cells and other eukaryotes
How does CRISPR do it?
One major restriction! Jinek, M., et al Science 337: 816 (2012)
CRISPR/Cas9 as a tool for genome editing CRISPR requires two parts: Cas9 nuclease The nuclease that will cut at the targeteddna site Guide RNA (grna) Forms a complex with Cas9 (RNP) Specifies where the DNA cutting shouldoccur Either a RNA duplex (2 RNAs) or one single long RNA
Background CRISPR/Cas9 for Genome Editing DNA repair pathways: Non-homologous end joining (NHEJ) Error prone repair Creates insertions/deletions Homology-directed repair (HDR) Error free Can be used for intentional insertions or mutations
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Options for the CRISPR grna
CRISPR/Cas9 using a single guide RNA (sgrna) Single guide RNAs (sgrna) One long RNA combining the crrna and tracrrna by the addition of a linker sequence Too long (~ 100+) for efficient or affordablerna synthesis In vitro transcription can be used
CRISPR/Cas9 using a 2-Part guide RNA (grna) 2-Part crrna:tracrrna Complex Two separate RNAs that include hybridization region for duplexing crrna includes ~20 base targeting region and 16 additional bases for duplexingwithtracrrna tracrrna forms a duplex with crrna and complex with Cas9 tracrrna sequence does not change for different targets, only the crrna sequence changes
Using CRISPR CRISPR can be used in many ways Continuous cell culture Primary cell culture Animal model generation NGS sample preparation Test tube DNA digestions Generally, the delivery is the only component of the process that changes
Many future scenarios
gblocks Gene Fragments for CRISPR-Cas9 gene editing gblocks Gene Fragments have been used in numerous published CRISPR studies: Generating Cas9 and sgrna vectors Transfecting sgrna expression cassettes under U6 promoter control as described by the Church Lab (Harvard Medical School, Boston MA) Citations at www.idtdna.com/gblocks 26
What are gblocks? Double-stranded linear synthetic DNA fragments Sequence verified 200 ng delivered (for 500 bp) Affordable for research needs For all purposes that require dsdna 28
Established and preferred for genome editing grna backbone Current Protocols in Molecular Biology (2014), 31.1.1.-31.1.17 29
Complexed RNA oligonucleotides as the CRISPR grna
Both the crrna and the tracrrna can be truncated 1. Short/Short Better Worse when too short 2. Long/Short 3. Short/Long 4. Long/Long Worse 31
Verify editing using T7 Endonuclease I (T7EI) assay 1. PCR amplify region flanking the CRISPR site (400 1000 base amplicons) Heat, cool to form heteroduplexes 2. Incubate with T7EI (New England BioLabs) 3. Run on gel or Fragment Analyzer (Advanced Analytical) to visualize cleavage at heteroduplex mismatch sites 32
IDT CRISPR gene editing and mutation detection workflow Transfect 2 part RNA at 30 nm or gblocks fragment at 3 nm into Cas9 expressing cells Extract gdna after 48 hr with QuickExtract DNA Solution Heat gdna extract at 65 C for 15 min followed by 95 C for 15 min Amplify gdna with KAPA HiFi Polymerase and PCR assay targeting region of interest Add NEB buffer 2 to PCR, heat to 95 C and slowly cool to allow heteroduplex formation Digest heteroduplexes with 2 units of T7EI at 37 C for 1 hr Analyze digestion on Fragment Analyzer Electropherogramand peak table of separated sample on Fragment Analyzer 52% T7 cleavage The Fragment Analyzer (Advanced Analytical) provides reliable quantification of T7EI heteroduplex cleavage assay with 96-channel CE High resolution analysis of fragments 10 40,000 bp Rapid 1 hr run 1/10 th amount of DNA required to visualize
CRISPR gblocks Gene Fragment in HPRT gene (HEK293 Cas9 cells) 38094 S 38095 S 38115 S 38129 S 38231 S 38239 S 38256 S 38338 S 38371 S 38448 S 38478 S 38509 S 38510 S 38574 38626 S S + 2% Agarose gel Fragment Analyzer 23% 46% 43% 21% 0% 31% 27% 3% 47% 0% 41% 14% 39% 4% 36% + Note: sequence analysis shows 30% cleavage in T7EI assay = 60 70% total editing
T7EI cleavage (%) 100 90 80 70 60 50 40 30 20 10 Optimized 2-part CRISPR RNAs are superior to other grnas CRISPR grna Comparison 12 grnas Targeting HPRT (HEK293 Cas9 Cells) 2 part RNA (36/67) Native RNA (42/89) In vitro transcribed sgrna sgrna Expression Plasmid (2.7 kb) gblocks Gene Fragments sgrna HEK Cas9 cells 2 part RNA (30 nm) IVT RNA (30 nm) Plasmid (100 ng) gblocks Fragment (3 nm) 0 HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT 38094 S 38231 S 38371 S 38509 S 38574 S 38087 AS 38133 AS 38285 AS 38287 AS 38358 AS 38636 AS 38673 AS
T7EI cleavage (%) Activity of 2-part RNA vs in-vitro transcribed sgrnas 100 90 80 70 60 50 40 30 20 10 2 part RNA vs. IVT sgrna HPRT 12 sites (HEK293 Cas9 Cells) 2 part RNA IVT sgrna 0 HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT HPRT 38094 S 38231 S 38371 S 38509 S 38574 S 38087 AS 38133 AS 38285 AS 38287 AS 38358 AS 38636 AS 38673 AS
CRISPR on-target mutation profiles for varying RNA triggers Sanger sequencing of amplicons from one target site in HPRT as varying RNA triggers
Immune responses from in-vitro transcribed RNA IFITM1, RIGI, and OAS2 had similarly high induction when treated with in vitro transcribed sgrna (triphosphate removed) No inductions were detected when treated with 2-part RNA oligos
RNA triggers solved, what about the nuclease? Cas9 solutions from IDT 39
IDT Cas9 expression plasmid minimal vector Minimal vector (7.3 kb) Origin of replication Ampicillin resistance No selection marker Deliver Cas9 expression plasmid, followed by delivery of 2-part RNA Improvements in editing, other plasmid associated problems remain
CRISPR grna + Cas9 as ribonucleoprotein complex Simple, fast, and robust delivery Complex grna & Cas9 protein Deliver directly to cells using lipofection or electroporation Cas9 RNP = preferred method Protection of RNA reduced risk of degradation Higher editing compared to plasmid delivery No DNA present no integration events Tight control of Cas9 (on/off, nothing present in the cell that can make more) Reduced risk of mosaicism in animal embryo studies
S. p. Cas9 Nuclease 3NLS Recombinant Cas9 nuclease 1X N-terminal nuclear localization sequence (NLS) 2X C-terminal NLS Available in 100 µg and 500 µg amounts
Alt-R CRISPR-Cas9 System products from IDT Description Formats Alt-R CRISPR crrna Tubes: 2, 10 nmol; Plates: 2 nmol Alt-R CRISPR tracrrna 5, 20, 100 nmol Alt-R HPRT Positive Control Kits 2 nmol: human, rat and mouse Alt-R HPRT Positive Control crrna 2 nmol: human, rat and mouse Alt-R Negative Control crrna 3 sequence options Alt-R HPRT PCR Primer Mix 2 nmol each: human, rat and mouse Alt-R S.p. Cas9 Expression Plasmid Minimal plasmid (7 kb) Alt-R S.p. Cas9 3NLS nuclease 100 and 500 µg
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