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  • 1 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    PPR technology

    1

    4 ii

    PPR motif (35 aa) v t Y n t l I s g l c k a G r l e e A l e l f e e M k e k - G i a P d v 1 4 ii

    - A - G - U - C - A - C - U - G - A - G - A -

    PPR protein

    RNA

    Base

    Human mitochondrial RNA pol. Ringel et al., Nature (2011)

    FTN VNS VTD FPD(1,4,ii)

    Rec. base

    [Base recognition code]

    RNA/DNA ( )

    Nu

    ER

    mRNA

    Protein

    ???

    RNA virus

    mitochondria

    DNA ncRNA

    Pentatricopeptide repeat

  • 2 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    l Dh l / / :

    5 )

    l N (

    l N ( .

    l ( 2 R( / R( / R(

    l ( l 0

    A 1 N

    3 P5

  • 3 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    Vision ~ New Tools Lead to a New World~

    < A D >6 :C : 167: 5 D A < >

    N PR P ab _ JJ

    3CA :

    02.

    42.

    42.

    /:>> : ::C -

    /:>>

  • 4 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    3. Genome editing (since 2010) • ゲノムを自在に書き換える。

    Science Breakthrough of the year 2013, 2014, 2015

    Break-through technologies for the cell engineering

    NGS

    G.E.AI

    1K

    1M

    1G

    1T

    Pr oc

    es si

    ng s

    pe ed

    /d ay

    1985 1990 1995 2000 2005 2010

    ←Human Genome (3 G bp)

    Manual seq.

    ABI377

    ABI3700

    ABI3730

    GS20(454)

    GAI(Solexa)

    SOLiD

    SOLiD3

    Heliscope

    GAII(Solexa)

    FLX Titanium

    PacBio

    1. 大規模配列解析技術(since 2004) ヒトゲノム : 1日で解読

    • 多様な生物のゲノム情報を理解 • 病気などの異常によるゲノム情報変化を理解

    AlphaGo by Google DeepMind

    2. 人工知能 (since 2015) • ゲノム情報改変の理論を理解 • 遺伝子回路の構築原理を理解

  • 5 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    BA

    C

    B

    10

    0.5 1

    http://www.asahi.com/articles/ photo/AS20150326000124.html

    Genome editing ( )

  • 6 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAA TGCAGCTGTAATACGACTCACTATAGGGCAAGCTTAAAAAGCCTTCCATT TTCTATTTTGATTTGTAGAAAACTAGTGTGCTTGGGAGTCCCTGATGATT AAATAAACCAAGATTTTACCATGACTGCAATTTTAGAGAGACGCGAAAG CGAAAGCCTATGGGGTCGCTTCTGTAACTGGATAACTAGCACTGAAAAC CGTCTTTACATTGGATGGTTTGGTGTTTTGATGATCCCTACCATGGTGAG CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGA GGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCC ACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAA CTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG ACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGA GAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC ACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGAA TTCCAACTGAGCGCCGGTCGCTACCATTACCAACTTGTCTGGTGTCAAAA ATAATAGGCCTACTAGTCGGCCGTACGGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCG CGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCAT CAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCA CAGATGCGTAAGGAGAAAATACCGCATCAGGCGGCCTTAAGGGCCTCG TTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGA TAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTT CCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT

    DNA

    DNA 4 A, T, G, C)

    1 → 1/4

    3 → 1/4 1/4 1/4 (1/4n; n=3 1/64

    30 1

    1/416= 1/42

    →16

    DNA

    DNA

    =

  • 7 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    DNA

    * * * * A T T C

    * * * * A T T C

    * * * * A T T C

    * * * * A T T C

    DNA

    DNA

    ?

    =

  • 8 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    =

    DSB

    Donor DNA (~16 kb)

    Donor DNA (ssDNA, 100b)

    NHEJ HR

    DSB: double strand break NHEJ: non homologous end joining, HR: homologous recombination,

    **

    (no-GMO?) ZFN-1

    +

    (no-GMO?) ZFN-2

    (GMO) ZFN-3

  • 9 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    G i y M GF T F M G F ML E

    T v v E

    -

    P v E

    -r c

    c F ML E

    T t e lh a

    o s A

  • 10 Copyright 2019 c) by EditForce Inc. All Rights Reserved.

    p • i e • e

    S 2o P A

    a 2

    Val83, Ser85, Asp87 and Thr89), with each protomer contributing three residues. Because many of the interactions are redundant, recognition of A(–7) will be described in detail and only the differences found in the A+1 binding pocket will be highlighted. For clarity, the prime symbol denotes residues located on the opposite protomer of the PP7DFG dimer.

    The upper surface of the A(–7) binding pocket is formed by the hydrophobic side chain of Val83¢ and the aliphatic portion of the Lys58¢ side chain, which make van der Waals contacts with the base (Fig. 2b,c). In the A+1 pocket, the side chain amine of Lys58 hydrogen bonds to the 2¢OH of G+3 and likely stabilizes its C2¢-endo sugar pucker (Fig. 2c). Asp87, Thr89 and Ser85¢ form the middle level of the pocket and make sequence-specific contacts to the adenine base. The side chain OH of Thr89 is within hydrogen-bonding distance of both the adenine N7 and N6 exocyclic amines. The backbone carbonyl of Asp87 is also within hydrogen-bonding distance of the N6 exocyclic amine. The third RNA-protein interaction at this level is a hydrogen bond between the OH of Ser85¢ and adenine N1. Arg54, whose guanidinium group makes a cation-p stacking interaction with the adenine base, forms the base of the binding pocket. The position of the Arg54 side chain is buttressed by hydrogen bonds to the side chain of Asp87. In the A+1 pocket, the Arg54¢ guanidinium group makes two hydrogen bonds to O6 and N7 of G+3 to specifically recognize the guanine base. (Fig. 2c). The importance of Lys58 and Arg54 in RNA recognition is supported by experiments that demonstrate that muta- tion of these residues leads to severe repression defects4.

    The PP7DFG–RNA complex is further stabilized by several hydro- gen bonds and electrostatic interactions outside of the adenine recognition pockets. The backbone amide of Gly48¢ and the side chain of Asn47¢ make hydrogen bonds to phosphate oxygens of A(–2) and U(–1), respectively. The U(–1) nucleotide is extended away from the loop into a pocket formed by Thr51¢, Ala52¢, Val91¢ and Thr81. The backbone amide and side chain OH of Thr81 make hydrogen bonds to the O2 and 2¢-OH of U(–1). Another potential hydrogen bond exists between the side chain carboxylic acid of Asp60 and the A+1 O2¢, which may stabilize its C2¢-endo sugar pucker. A similar interaction is observed in the MS2 coat protein–RNA complex, where Glu63 is hydrogen bonded to the U(–5) 2¢ OH13. Crystal-packing

    differences result in the side chain of Arg24 stacking with the guanine base of G+4 and contacting either its O4 or phosphate oxygens. Weak electron density and alternate conformations of the Arg24 side chain suggest that this interaction does not contribute substantially to the overall affinity of complex. There are also several positively charged residues (Arg24¢, Arg39, Arg45¢) that may participate in favorable electrostatic interactions with the phosphate backbone of the RNA.

    Although the PP7 and MS2 coat proteins share similar protein scaffolds, their RNA-binding surfaces have evolved to specifically recognize distinct RNA hairpins. The most notable difference between the two structures is the location of the adenine-recognition pockets, which are important components of binding for both coat pro- teins12,14,15. In the PP7 coat protein, the pockets are aligned along

    b c

    5′ 5′ 3′3′

    3′

    5′

    G - C

    A - U

    G - C

    A - U A - U

    C - G

    A

    A U G

    A U

    C - G

    G - C G - C

    G

    G - C

    G - C

    G - C

    U - A

    C - G A - U

    A - U

    A

    A U U

    A

    N

    N

    N N

    C

    C

    C C

    –14 –13

    –12

    –11

    –10 –9

    –8

    –7

    –6

    –5

    –4 –3

    –2 –1

    +1

    +2 +3

    +1 +2

    +3

    +4+5

    +6

    +7 +8

    +9

    +10 +11

    +12 +13

    –2

    –3 –4

    –5

    –6

    –7 –8

    –9 –10

    –11

    –12

    –1

    MS2 GA

    1 10 20 30 40 50 60 70 80 90 100 110 120

    Qb PP7

    a

    * * **

    +1

    3′5′

    +1 Figure 1 Coat-protein sequence alignment and overview of the MS2 coat protein and PP7DFG complexes with RNA. (a) Alignment of four ssRNA bacteriophage coat proteins. (b,c) MS2 coat protein–RNA (2BU1) (b) and PP7DFG–RNA (c) complexes are shown as cartoons. In both structures the RNA hairpin (orange, adenine; red, guanin