上原記念生命科学財団研究報告集, 34 (2020)ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧

40. ඃᛶኚ␗ࢆᣢࡘ㑇ఏᛶ⑌ᝈ࡟ᑐࡍࡿ᰿ᮏⓗ἞⒪ἲࡢ㛤Ⓨ㕥ᮌ ၨ୍㑻኱㜰኱Ꮫ ኱Ꮫ㝔ᇶ♏ᕤᏛ◊✲⛉ ≀㉁๰ᡂᑓᨷ ᶵ⬟≀㉁໬Ꮫ㡿ᇦKey words㸸ࢤࣀ࣒⦅㞟㸪HITI㸪SATI㸪ඃᛶ㑇ఏᏊኚ␗⥴ ゝඛኳⓗ࡞㑇ఏᏊኚ␗࡟ࡼࡾⓎ⑕ࡍࡿ㑇ఏ⑓࡜ࡋ࡚ 6,000 ௨ୖࡢ⑌ᝈྡࡀࡍ࡛࡟ሗ࿌ࡉࢀ࡚࠾ࡾࠊ10,000 ௨ୖࡢ  㑇ఏᛶ⑌ᝈࡀᏑᅾࡍࡿ࡜᥎ ࡉࢀ࡚࠸ࡿࠋ୍᪉࡛ࠊࡑࡢ࡯࡜ࢇ࡝ࡀ᰿἞ࡍࡿྍ⬟ᛶࡀ࡞ࡃ᭷ຠ࡞἞⒪ἲࡀᏑᅾࡋ࡞࠸
㞴἞ᛶ㑇ఏ⑓࡛࠶ࡿࠋ㏆ᖺࠊࢤࣀ࣒ࡢᶆⓗ㓄ิࡢࡳࢆ≉␗ⓗ࡟ษ᩿࣭ᨵኚࡍࡿࢱࣥࣃࢡ㉁ࠗேᕤ DNA ษ᩿㓝⣲࠘ࡀ 㛤Ⓨࡉࢀࠊᶆⓗ㑇ఏᏊࡢ◚ቯ㸦㑇ఏᏊࣀࢵࢡ࢔࢘ࢺ㸧ࡸࠊእ࠿ࡽࡢ㑇ఏᏊᤄධ㸦㑇ఏᏊࣀࢵࢡ࢖ࣥࡸ㑇ఏᏊಟ᚟㸧࡞࡝ࠊᵝࠎ࡞⣽⬊✀࣭⏕≀✀࡛⮬⏤⮬ᅾ࡟ࢤࣀ࣒㓄ิࢆࢹࢨ࢖࣭ࣥᨵኚࡍࡿࠗࢤࣀ࣒⦅㞟࠘ᢏ⾡ࡀ㛤Ⓨࡉࢀ࡚ࡁࡓࠋ≉࡟   ㏆ᖺ࡛ࡣࠊCRISPR/Cas9 ࡢ㛤Ⓨ࡟ࡼࡗ࡚ࢤࣀ࣒ DNA ࡢ௵ពࡢ㒊఩࡟ษ᩿ࢆᐜ᫆࡟ຍ࠼ࡿࡇ࡜ࡀྍ⬟࡜࡞ࡗࡓࡀࠊ ษ᩿㒊఩࡬ࡢ㑇ఏᏊࣀࢵࢡ࢖ࣥࡸ㑇ఏᏊ⨨᥮࡟ࡣࠊ⣽⬊ࡀ᭷ࡍࡿ DNA ಟ᚟ᶵᵓࡢ୍✀࡛࠶ࡿࠗ┦ྠ⤌᥮࠼ಟ᚟࠘ࡀ ⏝࠸ࡽࢀ࡚ࡁࡓࠋࡋ࠿ࡋࠊ┦ྠ⤌᥮࠼ಟ᚟࡟ࡼࡿࢤࣀ࣒⦅㞟࡟ࡣ㧗࠸⣽⬊ศ⿣άᛶࡀᚲせ࡛࠶ࡿࡓࡵࠊ⏕⌮ⓗ࡟⣽⬊ ศ⿣ࢆ⾜ࡗ࡚࠸࡞࠸࡯࡜ࢇ࡝ࡢ⏕యෆࡢ⣽⬊࡬ࡢᛂ⏝ࡣ㠀ᖖ࡟ᅔ㞴࡛࠶ࡗࡓࠋࡇࡢࡼ࠺࡞⫼ᬒࡢ୰ࠊ➹⪅ࡣ㠀ศ⿣⣽⬊࡛ࡶάᛶࡢ࠶ࡿࠕ㠀┦ྠᮎ➃⤖ྜࠖ⤒㊰ࢆ฼⏝ࡋࡓᶆⓗ㑇ఏᏊࡢᨵኚᡭἲࢆ⊂⮬࡟㛤Ⓨࡋࠊ⏕యෆࠊ≉࡟㠀ศ⿣⣽⬊࡛࠶ࡿ⚄⤒ࠊ➽⫗ࠊ⥙⭷࡟࠾ࡅࡿᶆⓗࢤࣀ࣒㓄ิࢆ⮬⏤࡟ᨵኚࡍࡿୡ⏺ึࡢᢏ⾡ࢆ㛤ⓎࡋࠊHITI㸦Homology-IndependentTargeted Integration㸧࡜ྡ௜ࡅࠊᐇ㝿࡟㑇ఏᛶ⑌ᝈ࡛࠶ࡿ⥙⭷Ⰽ⣲ኚᛶ⑕ࡢࣔࢹࣝࣛࢵࢺ࡟ᑐࡋ࡚どぬᶵ⬟㞀ᐖࡢ ἞⒪ຠᯝࡀᚓࡽࢀࡓ㹙1㹛ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ᪤Ꮡࡢ HITI ἲ࡛ࡣࠊ௵ពࡢ㓄ิࢆࢤࣀ࣒ᶆⓗ㒊఩࡟ᤄධࡍࡿࡇ࡜ࡣ࡛ࡁ ࡚ࡶཎᅉኚ␗ࢆྲྀࡾ㝖ࡃࡇ࡜ࡣฟ᮶࡞࠸࡜࠸࠺኱ࡁ࡞ၥ㢟Ⅼࡀ࠶ࡾࠊ἞⒪ྍ⬟࡞ᶆⓗࡣ㑇ఏᏊࡢ୍㒊ࡀḞⴠࡋ࡚࠸ࡿḞኻኚ␗ࡢࡳ࡛࠶ࡗࡓࠋᮏ◊✲࡛ࡣࠊ⮬ࡽ㛤Ⓨࡋࡓ HITI ᢏ⾡ࢆᨵⰋࡍࡿࡇ࡜࡛ࠊᚑ᮶ࡢ㑇ఏᏊ἞⒪ἲࡢ἞⒪ᑐ㇟࡜ ࡞ࡽ࡞࠸ࠗGain of function㸦ඃᛶ㑇ఏᏊኚ㸧
࠘ࢆ἞⒪ྍ⬟࡜ࡍࡿ᪂つࢤࣀ࣒⦅㞟἞⒪ἲࡢ㛤Ⓨࢆ⾜࠸ࠊඃᛶኚ␗ࢆ᭷ࡋ⑓ⓗ࡟⪁໬ࡀಁ㐍ࡍࡿ᪩⪁⑕㸦ࣉࣟࢪ࢙ࣜ࢔⑕ೃ⩌㸧࣐࢘ࢫ࡟࠾࠸࡚ࠊ඲㌟ᛶࡢࢤࣀ࣒⦅㞟἞⒪࡟ᡂຌࡋࡓ㹙2㹛ࠋ᪉ ἲ1㸬 ᪂つࢤࣀ࣒⦅㞟ᢏ⾡ࡢ㛤Ⓨ➹⪅ࡀ㛤Ⓨࡋࡓ㠀┦ྠᮎ➃⤖ྜάᛶࢆ฼⏝ࡋࡓ HITI ᢏ⾡ࡢ≉ᚩ࡜ࡋ࡚ࠊ௵ពࡢ㓄ิࢆࢤࣀ࣒ᶆⓗ㒊఩࡟ᤄධࡍࡿࡇ࡜ࡀᣲࡆࡽࢀࡿࡀࠊᮏ◊✲࡛ࡣࡇࡢ≉ᚩࢆ⏕࠿ࡋࡓඃᛶኚ␗ࡢ἞⒪ἲࡢ㛤ⓎࢆⓎ᱌࣭ᐇ⾜ࡍࡿࠋලయⓗ࡟ࡣࠊඃᛶ ኚ␗ࢆᣢࡘ࢚ࢡࢯࣥࡢ๓ࡢ࢖ࣥࢺࣟࣥ࡟௨㝆ࡢ࢚ࢡࢯࣥ࡜࢖ࣥࢺࣟࣥ࡜ 3̓UTR ࢆ⤖ྜࡋࡓࠗmini-gene࠘ࢆ HITI ἲ࡟ࡼࡾᤄධࡍࡿࠗ࢖ࣥࢺࣟࣥࣀࢵࢡ࢖ࣥἲ㸦ᅗ 1㸧࠘ࢆ⪃᱌ࡋࠊࡑࡢ᭷ຠᛶࢆ᳨ウࡋࡓࠋ2㸬 ࣉࣟࢪ࢙ࣜ࢔⑕ೃ⩌ࣔࢹ࣐ࣝ࢘ࢫࢆ⏝࠸ࡓ἞⒪ຠᯝࡢ᳨ウ᪂つࢤࣀ࣒⦅㞟἞⒪ἲࡢ᭷ຠᛶࢆ᳨ウࡍࡿࡓࡵࠊ⏕యෆ࡛ࡢ DNA ᑟධ࡟ඃࢀ࡚࠸ࡿ࢔ࢹࣀ⬻㝶క࢘࢖ࣝࢫ㸦AAV㸧࣋ࢡࢱ࣮࡟㑇ఏᏊ἞⒪⏝ DNA ࢆᦚ㍕ࡋࡓࠋࡇࢀࢆ⑓ⓗ࡟⪁໬ࡀಁ㐍ࡍࡿ᪩⪁⑕࣐࢘ࢫ࡟㟼⬦ὀᑕࡋ඲㌟ᛶ࡛ࢤࣀ࣒⦅㞟἞⒪ࢆヨࡳࡓࠋ1ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌ᅗ 1㸬࢖ࣥࢺࣟࣥࣀࢵࢡ࢖ࣥἲ
ඃᛶኚ␗ࡢᣢࡘ࢚ࢡࢯࣥࡢ๓ࡢ࢖ࣥࢺࣟࣥ࡟௨㝆ࡢ࢚ࢡࢯࣥ+࢖ࣥࢺࣟࣥ+3̓UTR ࢆᤄධࡍࡿࡇ࡜࡛ ṇᖖᆺࡢ㑇ఏᏊࡀⓎ⌧ࡍࡿࠋኚ␗ࢆྲྀࡾ㝖ࡃࡇ࡜ࡢฟ᮶࡞࠸ᚑ᮶ࡢ HITI ἲࡢḞⅬࢆ⿵࠺㑇ఏᏊಟ᚟ἲ࡛࠶ࡿࠋ⤖ ᯝ1㸬 ᪂つࢤࣀ࣒⦅㞟ᢏ⾡ࡢ㛤Ⓨ⪃᱌ࡋࡓ࢖ࣥࢺࣟࣥࣀࢵࢡ࢖ࣥἲࡣࠊᅗ 1 ࡢࡼ࠺࡟ࢤࣀ࣒୰ࡢ┦ྠ㒊఩࡟ᑐࡋ࡚ࢻࢼ࣮DNA ࡢ∦ഃࡢࡳ┦ྠ㓄ิ
㸦one arm㸧ࢆᣢࡘࡀࠊࢤࣀ࣒ᶆⓗ㒊఩࡜ࢻࢼ࣮DNA ࢆ⣽⬊ෆ࡛ྠ᫬࡟ษ᩿ࡍࡿࡇ࡜࡛ࠊእ᮶㑇ఏᏊࡀ∦ഃࡢࡳࡢ ┦ྠ㓄ิࢆ௓ࡋ࡚ᶆⓗ㑇ఏᏊ࡟⨨᥮ࡉࢀࡿ᪂つ㑇ఏᏊࣀࢵࢡ࢖ࣥ⤒㊰ࢆⓎぢࡋࠊࠕone-armed HDR㸦oaHDR㸧ࠖ࡜  ྡ௜ࡅࡓ㸦ᅗ 2㸧ࠋࡇࡢᢏ⾡ࡣ➹⪅ࡽࡀ௨๓㛤Ⓨࡋࡓ HITI ἲ࡛ࡣ୙ྍ⬟࡛࠶ࡗࡓ㓄ิ⨨᥮ࡶ⾜࡞࠼ࡿࡓࡵࠊࡼࡾỗ⏝ᛶࡢ㧗࠸ᢏ⾡࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࠊ᪂つࡢࢤࣀ࣒⦅㞟ᢏ⾡ࠕSATI ἲ: intercellular linearized Single homology Armdonor mediated intron-Targeting Integrationࠖ࡜ྡ௜ࡅࡓࠋ᭦࡟ oaHDR ࡢཎ⌮ࢆヲ⣽࡟ㄪ࡭ࡿࡓࡵࠊshRNA ࢆ⏝࠸࡚ DSB ಟ᚟⤒㊰࡟㛵୚ࡍࡿ㑇ఏᏊࢆⓎ⌧ᢚไࡋࡓ㸦ᅗ 3㸧ࠋࡇࡢ⤖ᯝࠊศ⿣⣽⬊࡛୺࡞ DSB ಟ᚟⤒㊰࡛࠶ࡿ HDR ࡜NHEJ ࡢ୧᪉ࡢ㑇ఏᏊ⩌ࡀ㛵ࢃࡿ」ྜⓗ࡞᪂つ DSB ಟ᚟⤒㊰ࡀ⚄⤒⣽⬊࡛ࡣാ࠸࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ2ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌ᅗ 2㸬oaHDR ࡜ NHEJ ࢆ฼⏝ࡋࡓ SATI ἲኚ␗ࡢᏑᅾࡍࡿ࢚ࢡࢯࣥࡼࡾୖὶࡢ࢖ࣥࢺࣟࣥ࡟ࠊኚ␗ࢆᣢࡓ࡞࠸㑇ఏᏊࡢ୍㒊ࢆᣢࡓࡏࡓእ᮶ DNA
ࢆࣀࢵࢡ࢖ࣥࡍࡿࠋࢻࢼ࣮DNA ࡀ᭷ࡍࡿ∦ഃࡢ࣍ࣔࣟࢪ࣮࢔࣮࣒ࢆ௓ࡋࡓ᪂つ┦ྠ⤌᥮࠼⤒㊰ࢆⓎぢࡋࠊoaHDR ࡜ྡ௜ࡅࡓࠋSATI ἲࡣ oaHDR ࠿ NHEJ㸦HITI㸧ࡢ࡝ࡕࡽࡢ⤒㊰࡛ᤄධࡉࢀ࡚ࡶṇᖖᆺࢱࣥࣃࢡ㉁ࡀⓎ⌧ࡍࡿࡓࡵࠊඃᛶ✺↛ኚ␗ࢆྵࡴ࡯࡜ࢇ࡝ࡢኚ␗ࡀಟ᚟ᑐ㇟࡜࡞ࡿࠋ

3ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧
㻌Sub pathway
Initial DSB resection
NHEJGeneRad50Ku70/Xrcc6
Ku80/Xrcc5
Lig3
Xrcc1
Rad51
Rad52
Ercc1
Ercc4/Xpf
Smc5
Rad18
Msh2
Apex1

alt-NHEJ
HDR
SSA

Cohesin formation
Upstream activator
Mismatch repair
Base-excision repair

**
***

**
****

0.5

**

****

***

****

*
*
*

*

**

Relative percentage of inhibition
GFP+ cells over transfected cells

1

**bb㻌

Clone ID (Sigma)
TRCN0000336380
TRCN0000321228
TRCN0000312877
TRCN0000070980
TRCN0000077239
TRCN0000012660
TRCN0000233363
TRCN0000238086
TRCN0000175845
TRCN0000241751
TRCN0000124781
TRCN0000042496
TRCN0000304312****Repair pathway
DSB repair

Ercc1

aErcc4/Xpfa㻌

Homology

DSB Induction
by CRISPR/Cas9

3

43’UTRgRNA target

X

X

4

GOI 3’UTR

1

2

SATI donor

1

2

3

Ku70-Ku80
heterodimer

Base excision repair

Mismatch repaircTargetagene locus

Apex1

Msh2

Smc5

Cohesinformation
Upstream
activation

HDR SSA

Rad18

Rad52

Rad51

Xrcc1

Lig3

Ku80/Xrcc5

DSBrepairDNA repair
pathway:c㻌

alt-NHEJ

Subpathway:

Ku70/Xrcc6Rad50
Initial DSB
resection

Gene:NHEJ

Control

043’UTR

4GOI 3’UTR(One-armed donor)
NHEJ

End resection?

Ku70 / Ku80

Mre11-Rad50-Nbs1
Rad51
121343’UTR
3UT

4GOI 3’UTR

2344

GOI 3’UTR

3’UTR

HITI
ΔRad51

Single-strand annealing?

213321G O I

434

GOI 3’UTR

43’UTR

Lig333’UTR
3’UTR

43’UTR

Large deletion

oaHDR (One-armed HDR)

22

AltNHEJ?

4411124 3’UTR

GOI 3’UTRᅗ 3㸬oaHDR ࡢศᏊᶵᵓ
a㸧౑⏝ࡋࡓ shRNA ࣜࢫࢺࠋb㸧shRNA ࢆ⏝࠸ࡓ㑇ఏᏊࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿᇵ㣴⚄⤒⣽⬊࡛ࡢ㑇ఏᏊࣀࢵࢡ࢖ࣥຠ⋡ࡢ ᐃ
Student’s t-test㸸****P < 0.0001ࠊ***P < 0.001ࠊ** P < 0.01ࠊ* P < 0.05ࠋ
c㸧oaHDR ࡢศᏊᶵᵓࣔࢹࣝࠋ

4ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧
㻌2㸬 ࣉࣟࢪ࢙ࣜ࢔⑕ೃ⩌ࣔࢹ࣐ࣝ࢘ࢫࢆ⏝࠸ࡓ἞⒪ຠᯝࡢ᳨ウ⏕ᚋ 1 ᪥┠ࡢ᪩⪁⑕࣐࢘ࢫ࡟㟼⬦ࢆ௓ࡋ AAV ࢆὀධࡋࠊ඲㌟ᛶ࡛ࢤࣀ࣒⦅㞟἞⒪ࢆヨࡳࡓࠋ⤖ᯝ࡜ࡋ࡚ࠊ⪁໬ࡢ ⾲⌧ᆺࡢ⦆࿴ࡸࠊ▷⦰ࡋࡓᑑ࿨ࢆ 1.5 ಸᘏ㛗࡛ࡁࡿࡇ࡜ࢆ☜ㄆ࡛ࡁࠊᙜヱᢏ⾡ࡣࢤࣀ࣒⦅㞟἞⒪ἲ࡜ࡋ࡚᭷ຠ࡞ᡭẁ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸦ᅗ 4㸧ࠋ୍㐃ࡢ◊✲ᡂᯝࡣ 2019 ᖺࡢ Cell Research ㄅ࡟ᥖ㍕ࡉࢀࡓ㹙2㹛ࠋa య㛗b᪩⪁⑓䝰䝕䝹䝬䜴䝇䛾ᑑ࿨ᘏ㛗ຠᯝ㔝⏕ᆺ
SATI἞⒪ᚋ

἞⒪䛺䛧
἞⒪䛺䛧
SATI἞⒪ᚋ᪩⪁⑓䝰䝕䝹䝬䜴䝇ᅗ 4㸬ඃᛶኚ␗ࢆᣢࡘࣉࣟࢪ࢙ࣜ࢔⑕ೃ⩌ࣔࢹ࣐ࣝ࢘ࢫࡢࢤࣀ࣒⦅㞟἞⒪ᅗ㻠㻌㻌ඃᛶኚ␗䜢ᣢ䛴䝥䝻䝆䜵䝸䜰⑕ೃ⩌䝰䝕䝹䝬䜴䝇䛾䝀䝜䝮⦅㞟἞⒪SATI἞⒪࡟ࡼࡿ᪩⪁⑕ࣔࢹ࣐ࣝ࢘ࢫࡢ㑇ఏᏊ἞⒪ࠋ⏕ᚋ 118 ᪥ࡢ᪩⪁⑕ࣔࢹ࣐ࣝ࢘ࢫ࡛ぢࡽࢀࡿ⪁໬⑕≧㸦aࠊయ㛗㸧ࡸ⏕Ꮡ⋡㸦b㸧ࡢ㒊ศᅇ᚟⪃ ᐹࡇࡢࡼ࠺࡟ᮏ◊✲࡛ࡣࠊᚑ᮶ࡢ㑇ఏᏊ἞⒪ἲ࡛ࡣ἞⒪ࡀᅔ㞴࡛࠶ࡗࡓඃᛶ✺↛ኚ␗ࢆ᰿ᮏ࠿ࡽಟ᚟ྍ⬟࡞᪂つࢤࣀ࣒⦅㞟἞⒪ἲࡢ㛤Ⓨ࡟ᡂຌࡋࡓࠋ௒ᚋᮏ◊✲࡛㛤Ⓨࡉࢀࡓᢏ⾡ࡢᏳ඲ᛶࡀ☜ㄆࡉࢀࠊ㑇ఏᏊಟ᚟ຠ⋡ࡶ᭦࡟ᨵⰋࡉࢀࡿࡇ࡜࡛ࠊᡂேࡢ⚄⤒࣭ᚰ⮚࣭➽⫗࣭⥙⭷࡞࡝ᵝࠎ࡞⤌⧊ࡲࡓࡣ඲㌟࡟␗ᖖࢆᣢࡘᩘከࡃࡢ㞴἞ᛶ㑇ఏ⑓࡟ᑐࡋࠊࡑࡢ  ཎᅉ࡜࡞ࡿ␗ᖖ㑇ఏᏊࢆ⑓ኚ㒊఩࡛┤᥋ಟ᚟ࡍࡿ་⒪࡬ࡢᛂ⏝࡬⧅ࡀࡿࡇ࡜ࢆᮇᚅࡋ࡚࠸ࡿࠋඹྠ◊✲⪅࣭ㅰ㎡ᮏ◊✲ࡣࠊ⡿ᅜࢯ࣮ࢡ⏕≀Ꮫ◊✲ᡤ࣋ࣝࣔࣥࢸᩍᤵࢆጞࡵከࡃࡢඹྠ◊✲⪅࡟ཧ⏬࠸ࡓࡔ࠸ࡓࠋࡲࡓࠊᮏ◊✲ࡣ  බ┈㈈ᅋἲேୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋࡢ◊✲ຓᡂ㔠࡟ࡼࡗ࡚㐙⾜ࡉࢀࡓࠋࡇࡢሙࢆ೉ࡾ࡚ឤㅰ⏦ࡋୖࡆࡲࡍࠋᩥ ⊩
1㸧 Suzuki, K., Tsunekawa, Y., Hernandez-Benitez, R., Wu, J., Zhu, J, Kim, J. E., Hatanaka, F., Yamamoto, M.,Araoka, T., Li, Z., Kurita, M., Hishida, T., Li, M., Aizawa, E., Guo, S., Chen, S., Goebl, A., Soligalla, R.D., Qu, J.,
Jiang, T., Fu, X., Jafari, M., Esteban, C.R., Berggren, T., Lajara, J., Nuñez, E., Guillen, P., Campistol, J.M.,
Matsuzaki, F., Liu, G.H., Magistretti, P., Zhang, K., Callaway, E.M., Zhang, K. and Izpisua Belmonte, J.C. In
vivo genome editing via CRISPR-Cas9 mediated homology-independent targeted integration. Nature. 2016
Dec 1;540(7631):144-149. Epub 2016 Nov 16. PMID: 27851729 DOI: 10.1038/nature20565
2㸧 Suzuki, K., Yamamoto, M., Hernandez-Benitez, R., Li, Z., Wei, C., Soligalla, R.D., Aizawa, E., Hatanaka, F.,
Kurita, M., Reddy, P., Ocampo, A., Hishida, T., Sakurai, M., Nemeth, A.N., Nuñez Delicado , E., Campistol, J.M.,
Magistretti, P., Guillen, P., Esteban, C.R., Gong, J., Yuan, Y., Gu, Y, Liu, G.H., López-Otín, C., Wu, J., Zhang, K.
and Izpisua Belmonte, J.C. Precise in vivo genome editing via single homology arm donor mediated introntargeting gene integration for genetic disease correction. Cell Res. 2019 Oct;29(10):804-819. Epub 2019 Aug 23.
PMID: 31444470 DOI: 10.1038/s41422-019-0213-0
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