New RNA tools for optimized CRISPR/Cas9 genome editing

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  • Ashley Jacobi, Research ScientistIntegrated DNA Technologies

    New RNA tools for optimized CRISPR/Cas9 genome editing

    October 7th, 2015

    1

  • Implementing CRISPR/Cas9 gene editing

    2

  • Options for the CRISPR gRNA

    3

  • Repair of double-stranded breaksHR vs. NHEJ

    4

  • S. pyogenes Cas9 is a large protein, 1368 aa = 4104 bp

    Plasmid containing Cas9: 710 kb

    Transfection of a large plasmid results in variable and low transfection efficiency, making large quantitative comparison studies difficult

    Delivery of a Cas9 + sgRNA expression plasmid is difficult

    5

    Delivering large Cas9 expression plasmid to cells can be difficult

  • Optimizing CRISPR gRNA using HEK293-Cas9 cell line

    6

    Low, constant level of Cas9 present in HEK293-Cas9 Note the extremely high levels of Cas9 present in

    just a small fraction (~10%) of transfected cells using plasmid. Can this contribute to OTEs?HEK293-Cas9 Cells

    Western blotCas9 primary antibody

  • T7EI mismatch detection to assay gene disruption1. Transfect HEK-Cas9 cells with the CRISPR gRNA

    Alternatively deliver Cas9 as plasmid, mRNA or protein

    2. Incubate 48 hours, then harvest genomic DNA3. PCR amplify region around CRISPR site (4001000 base amplicons)

    Heat, cool to form heteroduplexes

    4. Incubate with T7 Endonuclease I (T7EI, New England BioLabs) 5. Run on gel or Fragment Analyzer (Advanced Analytical) to visualize cleavage at heteroduplex mismatch sites

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  • IDT CRISPR gene editing and mutation detection workflow

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    The Fragment Analyzer (Advanced Analytical) provides reliable quantification of T7EI heteroduplex cleavage assay with 96-channel CE High resolution analysis of fragments 1040,000 bp Rapid 1 hr run 1/10th amount of DNA required to visualize

    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 65C for 15 min followed by 95C

    for 15 min

    Amplify gDNA with KAPA HiFi

    Polymerase and PCR assay targeting region of interest

    Add NEB buffer 2 to PCR, heat to 95C and slowly cool to allow heteroduplex

    formation

    Digest heteroduplexes

    with 2 units of T7EI at 37C for 1 hr

    Analyze digestion on Fragment Analyzer

    Electropherogramand peak table of separated sample on Fragment Analyzer

    52% T7 cleavage

  • Options for the CRISPR gRNA

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  • gBlocks Gene Fragments for CRISPR Inexpensive gene synthesis product with rapid delivery

    High quality double-stranded DNA fragments 1252000 bp in length Sequence verified

    CRISPR gBlocks Gene Fragment = 364 bp sgRNA expression cassette Comprised of a 265 bp U6 promoter that drives transcription of a 99 base sgRNA

    www.idtdna.com/CRISPR

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    AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT

  • gBlocks Gene Fragments as sgRNA (three methods)

    1. Clone gBlocks Gene Fragments into an expression plasmid

    2. Use gBlocks Gene Fragment as template for in vitro transcribed sgRNA (IVT sgRNA)

    3. Directly transfect gBlocks Gene Fragment into cells without cloning

    11

    www.idtdna.com/CRISPR

    http://www.addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf

    gRNAbackbone

    Current Protocols in Molecular Biology (2014), 31.1.1-31.1.17.

    >14,000 gBlocks Gene Fragments manufactured for CRISPR

  • CRISPR gBlocks Gene Fragment in HPRT gene (HEK293 Cas9 cells)

    12

    38094S

    38095S

    38115S

    38129S

    38231S

    38239S

    38256S

    38338S

    38371S

    38448S

    38478S

    38509S

    38510S

    38574S

    38626S

    +

    23% 46% 21% 0% 31% 27% 3% 47% 0% 41% 14% 39% 4% 36%43%

    2% Agarose gel

    Fragment Analyzer

    Note: sequence analysis shows 30% cleavage in T7EI assay = 6070% total editing

    +

  • Validation of T7EI assay: % total editing compared to Sanger sequencing

    Sanger sequence analysis shows 30% cleavage in the T7EI assay = 6070% actual change at DNA level (T7EI misses small changes like single base indels)

    13

    Amplicons resulting in varying editing efficiencies via T7EI were cloned and sequenced.

    T7EI cleavage (%)

  • CRISPR gBlocks Gene Fragments sgRNA perform well across many sites(3 genes; 301 sites)

    14

    0102030405060708090

    100

    % Cleavage via 2U

    T7EI

    167 EMX1 Exon 3 (64% GC)

    91%*

    0102030405060708090

    100

    % Cleavage via 2U

    T7E1I

    92 STAT3 Exon 5/6 (47% GC)

    76%*

    0102030405060708090

    100

    % Cleavage via 2U

    T7EI

    42 HPRT Exon 7 (36% GC)

    81%*

    sgRNAs expressed from gBlocks Gene Fragments work well without the need to clone into plasmids

    Directly transfect into HEK-Cas9 cells at 3 nMEvery PAM site in 3 exons

    * Percentage of sgRNA designs with >20% editing efficiency by T7EI assay

    +

  • Options for the CRISPR gRNA

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  • Options for the CRISPR gRNA

    16

    UUAUAUCCAACACUUCGUGGUUUUAGA--GCUAG||||||| |||| A||||||| |||| A

    C-GGAAUAAAAUUGAACGAUAU| ||A| ||GUCCGUUAUCAACUUG

    |||| A|||| A

    AGCCACGGUGAAAG ||||||UCGGUGCUUU

    sgRNA: 99123 bases (99mer shown)

    20 base protospacer guide, target specific

    Near the length limit for chemical manufacturing Expensive to chemically make long sgRNAs for many sitesThis form is used in our gBlocks Gene Fragment sgRNAexpression cassette

    UUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCUGUUUUG||||||| ||||||||||||||

    C-GGAAUAAAAUUGAACGAUACGACAAAACUUACCAAGGUUGU| ||A| ||GUCCGUUAUCAACUUG

    |||| A|||| A

    AGCCACGGUGAAAG ||||||UCGGUGCUUUUUUU

    crRNA: 42 bases (target specific)tracrRNA: 89 bases (universal)

    42mer target specific (20 base target, 22 base constant) 89mer universal tracrRNAcan be made in bulk, making

    them more affordableCan these be optimized and shortened to improve function and lower cost?

  • Optimized length of crRNA and tracrRNA

    crRNA: 42 bases (20+22)tracrRNA: 89 bases (universal)

    UUAUAUCCAACACUUCGUGGUUUUAGA--GCUAUGCUGUUUUG||||||| ||||||||||||||

    C-GGAAUAAAAUUGAACGAUACGACAAAACUUACCAAGGUUGU| ||A| ||GUCCGUUAUCAACUUG

    |||| A|||| A

    AGCCACGGUGAAAG ||||||UCGGUGCUUUUUUU

    Native sequence

    Shorter

    Shorter

    Short

  • Both the crRNA and the tracrRNA can be truncated

    18

    1. Short/Short

    2. Long/Short

    3. Short/Long

    4. Long/Long Worse

    Worse when too short

    Better

  • 19

    0

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    90

    100

    89 nt tracrRNA 74 nt tracrRNA 70 nt tracrRNA 67 nt tracrRNA 65 nt tracrRNA 63 nt tracrRNA

    T7EI cleavage (%

    )

    42-nt crRNA39-nt crRNA36-nt crRNA34-nt crRNA

    Length optimization of crRNA & tracrRNAHPRT 38285 gRNA (HEK293 Cas9 Cells)

    Optimal length for crRNA is 36 nt; optimal tracrRNA is 67 nt

    +

  • 0

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    HPRT 38094 S

    HPRT 38231 S

    HPRT 38371 S

    HPRT 38509 S

    HPRT 38574 S

    HPRT 38087 AS

    HPRT 38133 AS

    HPRT 38285 AS

    HPRT 38287 AS

    HPRT 38358 AS

    HPRT 38636 AS

    HPRT 38673 AS

    T7EI cleavage (%

    )

    CRISPR gRNA Comparison12 gRNAs Targeting HPRT(HEK293-Cas9 Cells)

    2-part RNA (36/67)Native RNA (42/89)In vitro transcribed sgRNAsgRNA Expression Plasmid (2.7 kb)gBlocks Gene Fragments sgRNA

    Optimized 2-part CRISPR RNAs are superior to other gRNAs

    20

    HEK-Cas9 cells

    2-part RNA (30 nM)IVT RNA (30 nM)Plasmid (100 ng)

    gBlocks Fragment (3 nM)

    + or or or

  • Highly purified oligos are necessary for longer tracrRNAs

    21

    HEK-Cas9 cells

    2-part RNA, 30 nM

    Desalt crRNA

    Desalt vs. HPLC tracrRNA

    1. Shortened crRNA:tracrRNA performs better than native form2. 67mer tracrRNA functions well as desalted, shows slight improvement as HPLC3. Native 89mer tracrRNA requires highly purified synthesis

    0

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    20

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    HPRT 38094 S

    HPRT 38231 S

    HPRT 38371 S

    HPRT 38509 S

    HPRT 38574 S

    HPRT 38087 AS

    HPRT 38133 AS

    HPRT 38285 AS

    HPRT 38287 AS

    HPRT 38358 AS

    HPRT 38636 AS

    HPRT 38673 AS

    T7EI cleavage (%)

    HPLC vs. Desalted RNA Oligos HPRT 12 sites(HEK293 Cas9 cells)

    short crRNA (36nt) : short tracrRNA desalt (67nt)

    short crRNA (36nt) : short tracrRNA HPLC (67nt)

    long crRNA (42nt) : long tracrRNA desalt (89nt)

    long crRNA (42nt) : long tracrRNA HPLC (89nt)

    +

  • 2-part RNA oligos will be available from IDT this month! crRNA

    36 nt custom desalted RNA oligo Ability to order in 96-well plate format 2 or 10 nmol

    tracrRNA 67 nt HPLC purified RNA oligo Modified for nuclease stability 5, 20, or 100 nmol

    22

  • Be sure to check QC data on long synthetic RNAs

    23

    IDT tracrRNA Vendor X tracrRNA

    ESI-MS

  • 2-part RNA system functions well across many sites

    24All PAM sites in 6 exons, 553 sites (HEK293 Cas9 Cells)

    * **

    * * *

    * Percentage of sgRNA des