Optimized, chemically-modified crrna:tracrrna complexes for CRISPR gene editing Mark Behlke MD, PhD Chief Scientific Officer February 24, 2016 1
Implementing CRISPR/Cas9 gene editing 2
To focus on RNA triggers, produce a stable HEK293-Cas9 line Large Cas9 plasmids show low transfection efficiency that varies from day to day, making large quantitative comparison studies difficult. Low, constant level of Cas9 present in stable HEK293-Cas9 Note the extremely high levels of Cas9 present in just a small fraction of transfected cells using plasmid. Can this contribute to OTEs? 3
Detection of CRISPR editing by heteroduplex cleavage assay 1. Transfect HEK-Cas9 cells with trigger (2-part RNA, or gblocks Gene Fragments) Co-transfect Cas9 plasmid or mrna or protein if needed 2. Incubate 48 hr, then harvest DNA 3. PCR amplify region around CRISPR site (400 1000 bp amplicons) Heat and cool to form heteroduplexes 4. Incubate with T7EI or Surveyor mismatch endonuclease 5. Run on gel or Fragment Analyzer (Advanced Analytical) to visualize cleavage at heteroduplex mismatch sites 4
T7EI assay results for HPRT, CRISPR gblocks Gene Fragment walk 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 S 38626 S + HEK-Cas9 cell line 3 nm gblocks Fragment 48 hr time point 2% Agarose gel Fragment Analyzer 23% 46% 43% 21% 0% 31% 27% 3% 47% 0% 41% 14% 39% 4% 36% Note: 30% cleavage in the T7EI assay = ~60% actual change at DNA level 5
Options for the RNA triggers 6
gblocks Gene Fragments for CRISPR Inexpensive gene synthesis product with rapid delivery Identical to a PCR product suitable for cloning, amplification, or direct transfection into cells QC by Sanger sequencing and ESI-MS www.idtdna.com/gblocks CRISPR gblocks Gene Fragment = 364 bp sgrna expression cassette Comprised of a 265 bp U6 promoter that drives transcription of a 99 base sgrna AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATT TCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTAT TTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAG AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT 7
CRISPR gblocks Gene Fragments sgrna performance (3 genes; 301 sites) Guide sequences were made and tested for all PAM sites in 3 exons in 3 genes. sgrnas expressed from gblocks Gene Fragments work well without the need to clone into plasmids 8
Options for the RNA triggers 9
2-part crrna:tracrrna length optimization Native sequence Short Seed region NNNNNNNNNNNNNNNNNNNNGUUUUAGA--GCUAUGCUGUUUUG C-GGAAUAAAAUUGAACGAUACGACAAAACUUACCAAGGUUG U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUUUUUU crrna: 42 bases (20+22) tracrrna: 89 bases (universal) Shorter Shorter
T7EI cleavage (%) Optimal length for crrna is 36 nt; optimal tracrrna is 67 nt 100 90 80 70 60 50 40 30 20 10 Length optimization of crrna & tracrrna HPRT 38285 grna (HEK293 Cas9 Cells) 42-nt crrna 39-nt crrna 36-nt crrna 34-nt crrna 0 89 nt tracrrna 74 nt tracrrna 70 nt tracrrna 67 nt tracrrna 65 nt tracrrna 63 nt tracrrna 11
Deletion studies in the tracrrna GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU 67mer 62mer CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUA AGCAUAGCAAGUUAAAAUA GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AGCAUAGCAAGUUAAAAUAAGGCUAGUCC CCGAGUCGG AACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AACUUGAAAAAGUG CCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU GCACCGAGUCGGUGCU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU Deletions in the internal hairpin structures kill function of the tracrrna 12
Deletion of internal structure leads to loss of function GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUA AGCAUAGCAAGUUAAAAUA GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AGCAUAGCAAGUUAAAAUAAGGCUAGUCC CCGAGUCGG AACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AACUUGAAAAAGUG CCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU GCACCGAGUCGGUGCU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU CUUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCU C-GGAAUAAAAUUGAACGAUACGA U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUU Deletions in the internal hairpin structures kill function of the tracrrna 13
Deletion of internal structure leads to loss of function GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUA AGCAUAGCAAGUUAAAAUA GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AGCAUAGCAAGUUAAAAUAAGGCUAGUCC CCGAGUCGG AACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AACUUGAAAAAGUG CCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU GCACCGAGUCGGUGCU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU CUUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCU C-GGAAUAAAAUUGAACGAUACGA U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUU Deletions in the internal hairpin structures kill function of the tracrrna 14
Deletion of internal structure leads to loss of function GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUA AGCAUAGCAAGUUAAAAUA GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AGCAUAGCAAGUUAAAAUAAGGCUAGUCC CCGAGUCGG AACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AACUUGAAAAAGUG CCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU GCACCGAGUCGGUGCU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU CUUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCU C-GGAAUAAAAUUGAACGAUACGA U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUU Deletions in the internal hairpin structures kill function of the tracrrna 15
Deletion of internal structure leads to loss of function GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUA AGCAUAGCAAGUUAAAAUA GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGGUGCU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AGCAUAGCAAGUUAAAAUAAGGCUAGUCC CCGAGUCGG AACUUGAAAAAGUGGCACCGAGUCGGUGCU AACUUGAAAAAGUGGCACCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCC AACUUGAAAAAGUG CCGAGUCGG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU GCACCGAGUCGGUGCU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU CUUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCU C-GGAAUAAAAUUGAACGAUACGA U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUU Deletions in the internal hairpin structures kill function of the tracrrna 16
T7EI cleavage (%) Comparison of IDT vs. native 2-part crrna:tracrrna (12 HPRT sites) HEK-Cas9 cells 2-part RNA, 30 nm Desalt crrna HPLC tracrrna 17
T7EI cleavage (%) T7EI cleavage (%) T7EI cleavage (%) T7EI cleavage (%) T7EI cleavage (%) T7EI cleavage (%) gblocks Gene fragment sgrnas vs. 2-part Alt-R CRISPR RNA oligos Important to note: sgrna and 2-part systems do not always show optimal activity at the same sites gblocks fragment expressed sgrnas 3 nm 2-part Alt-R RNAs 30 nm HEK-Cas9 cells 18
Can chemical modification improve function of CRISPR RNAs? Of course yes! Oligo 101 unmodified nucleic acids are degraded by serum and/or cellular nucleases. Oligos need chemical modification to stabilize the nucleic acid and to help evade triggering the innate immune system. Long history of medicinal chemistry to find optimal approach for oligo modification for different applications which modifications to use vary with interaction with protein effector molecules (RNase H1, Ago2, Cas9 ) Pretty basic toolbox: PS bonds, 2 OMe, 2 F, LNA, end-blocking groups, etc. 19
The benefit of limited chemical modification was recently demonstrated for chemically synthesized sgrnas and truncated crrnas 20
Studying chemical modifications with over 400 RNA oligos CGGAAUAAAAUUGAACGAUACGA 5 U A GUCCGUUAUCAACUUG A A AGCCACGGUGAAA G UCGGUGCUUU 3 67mer tracrrna 5 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCU 3 20 base protospacer guide domain 16 base tracrrna binding domain 36mer crrna 67 bases 66 linkages 2 ends 135 places to modify 36 bases 35 linkages 2 ends 73 places to modify 21
Modification walk in the tracrrna Activity (T7EI assay) AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U A*G*CAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U Black = RNA Red = 2 OMe RNA * = PS bonds 22
Modification walk in the crrna Activity (T7EI assay) Black = RNA Red = 2 OMe RNA Green = 2 F RNA * = PS bonds Protospacer tracrrna-binding CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU CUUAUAUCCAACACUUCGUGGUUUUAGAGCUAUGCU 23
What we learned from the modification walks tracrrna cggaauaaaauugaacgauac*g*a 5 u A guccguuaucaacuug A A AGCCACGGUGAAA G UCGGUGCU*U*U 3 agcu = RNA AGCU = 2 OMe RNA * = PS bond 5 C*U*U*AUAUCCAACACuuCGuGGuuuUAGAGCUAU*G*C*U 3 20 base protospacer guide domain 16 base tracrrna binding domain = Major loss of function with 2 -mod = Minor loss of function with 2 -mod = Loss of function varies with sequence crrna 24
Results from beta test site customer investigating gene editing in activated human T-cells Human primary CD3+ T-cells activated with CD3/CD28 beads for 72 hr Target: TCRA Assay: loss of CD3 expression on FACS Washed, then transfected using Neon Electroporation System in 10 ml volume (Boissel et al., NAR 2013 protocol) Cas9 mrna 0.1 mg/ml + 10 mm crrna:tracrrna complex (optimized) Cas9 protein RNP 1:1 with crrna:tracrrna complex at 1 mm (2 4 mm better) 72 hr FACS with CD3 staining 25
tracrrna: crrna: unmod unmod unmod mod mod unmod mod mod neg con Cas9 mrna Cas9 RNP CD3 expression Chemical modification is important with Cas9 mrna, need both modified Chemical modification is helpful with Cas9 RNP, especially tracrrna 26
Utility in Zebrafish embryo microinjection crrna:tracrrna complexed with Cas9 protein Microinjected into Zebrafish embryos as RNP complex In this system, unmodified and modified RNAs performed equally well James Gagnon Alex Schier Harvard University 27
Utility in C. elegans microinjection Microinjection into C. elegans with Cas9 protein as RNP Modified tracrrna paired with unmodified or modified Dpy-10 crrnas Assess rate at which progeny are produced with Dpy-10 phenotype (dumpy, left roller) Brian Kraemer University of Washington, Seattle 28
Advantages of Alt-R CRISPR-Cas9 System, 2-part RNA Compared to IVT sgrnas, Alt-R CRISPR RNA: Easy to order, no work to prepare Mass spec QC Any yield needed is available Chemically modified to reduce toxicity from innate immune stimulation and improved performance due to nuclease stability Compared to chemically synthesized sgrnas, Alt-R CRISPR RNA: Easier to manufacture Much less expensive 29
Conclusions 1. Alt-R Synthetic RNA oligos mimicking the natural 2-part CRISPR system (crrna:tracrrna complex) function well in mammalian cells when Cas9 is expressed in the target cell (constitutive or transient), when made as Cas9 mrna, or when precomplexed with Cas9 protein as a RNP. 2. Optimized, shortened crrna:tracrrna complex shows higher editing activity. 3. Modified tracrrna gives improved results with almost all systems. 4. Modified crrna gives improved results, particularly when the RNAs have to survive inside the cell while waiting for Cas9 protein to be expressed. 5. The use of Cas9 protein + crrna:tracrrna RNP complex works well, simplifies workflow, and enables use of less highly modified RNAs (protein RNP helps protect the transfected RNAs). 30
Thanks to the scientists who contributed to these studies Integrated DNA Technologies Ashley Jacobi Garrett Rettig Michael Collingwood Michael Christodoulou Mollie Tiernan Sarah Zeiner Kyle McQuisten 31