Aki Hayashi Feb. 26, 2019

Short-homology-mediated CRISPR/Cas9-based method in fission yeast
Hayashi & Tanaka 2019, G3 https://doi.org/10.1534/g3.118.200976

Two plasmids (Cas9 + gRNA)

Single plasmid (gRNA-Cas9)

Cas974 mvx GH'

gRNA-Cas9 74 mvx GH'VSDNA– zX6 +R+ zX 4 +RgRNA74 mvx GH'VSDNA– zX6 +R

J#pnn ~

YESpnn ~

YESpnn ~– zX2 +R

2 ~ 3 +R

2 ~ 3 +R6 n;F6

@4
PCR~|n;F@4PCR
~|1. gRNA
1-1. gRNAnEDPAML(Protospacer adjacent motifXNGG)y6 TrfoK% Uy^cl\I nDNA
LTOU_w&dX5’ n020 bpygRNAjdhEDexY*oPAMn>3 bp0m xfsXlxqb6nIbm*1y$ggRNA ByJ#exYrfgRNAEDnfsnweb{T WSCRISPRdirectUigRNALn By,?excj`iaxYweb{n,?-m98K
nL 500 bpy dXn52<yJ#dhAM.lkUS,?yPexY

1-2. gRNAn36!n;FC(n B`Zxo}jdhfLyb$gtnu40%n[GCNy$ggRNAyQbYrfPAMLnI mbnA/T L`ZxgRNAn ByQbY

1-3. gRNAn}y/)ex
gRNA - Fw

5’-caccXXXXXXXXXXXXXXXXXXXX-3’

gRNA - Rv

5’-aaacXXXXXXXXXXXXXXXXXXXX-3’(gRNAL20 bpn:BOU

PAM (NGG)osl[BbsI*="m~iaxv\m4 bp]xgRNA EDnfsnWeb{CRISPRdirect

https://crispr.dbcls.jp

E-CRISP

http://www.e-crisp.org/E-CRISP/designcrispr.html

Bähler lab genome Regulation

http://bahlerweb.cs.ucl.ac.uk/cgi-bin/crispr4p/webapp.py

2. gRNAo¢¡2-1. o}¢¡

T

CACCXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXCAAA*10 x annealing buffer

3 ulgRNA Fw (100 uM)

6 ulgRNA Rv (100 uM)

6 ul

dH2O

15 ul

/ 30 ul#k}¢¡|¢QMI;|¢ju£k+{PCR¢nzi
0eUPCRC>j}¢¡"{[`mYV95˚C j25bfogU$>sfpx bi1j30 O>cyVPCRC>{6Xf}¢¡ {6biu@XV¤ 95˚C 2 U –2˚CSP j25˚Csj ¢¡adyV*10 x annealing buffer: 0.1 M Tris pH7.5~8, 0.5 M NaCl, 10 mM EDTA%

2-2. BbsInvygRNA74oBbsI

5’..GAAGACNN↓..3’3’..CTTCTGNN(N)4↑ ..5’

Cas974{?A\w:Dbfp65˚Cj305bicyV
Cas9G{uhpmojUj^zq–80˚CncyV

T37˚Cj1 &L 5V
KX 5&LpH_yV

10 x buffer

10 ul

Plasmid (1 ~ug)

X ul

BbsI (NEB) or
BpiI (Thermo Fisher)

0.4 uldH2O(5~10 U/ ul)

Y ul /100 ul} ¢N*-j %{8FcyV
2 5jBbsI{/ady Q65˚Cj20 RV¢,)sfp:Dbi{cyV:Dopo~]^XftU3]]y]WyV– 80˚CjcyV =BEnvy E]*nmyp2-3. gRNAoro_nbicyV¢¡lo~¢¡ju6V(.<oLigation high ver. 2{6bfo'{9cV}¢bf{100 o1!JbiZyV
16˚Cj30

5VLigation high (1/2 volume of DNA)

1 ul

BbsI digested Plasmid (~10 ng/ul)

1 ul

1/100 diluted annealed oligo

1 ul/ 3 ul2-4. !IJx(PQ.
§¦¥ln*7¥¥¢we©5 ulx*750 ulx¥¥¢we¨c5s30[Flb42˚Cs30B¦mc5s2[FlrfLB-Amp£¦wDG&ic37˚Cs1]mc

2.5. gRNAx¤¦¥xAO(PQ. LB-Amps]lb DNACLmc Cas9U",q y65˚C s30<lrf #mcgRNAx-AOy3?w¦¥wprAOmgbgRNA=x¡tS ¦tx=dn¤¦PCRw-AOs~c

3. DNA
3. 1. 9>$/9. swi6



PAM: NGG
5’-GAGGCTATGAATACCTTTTGAAATGGGAAGGTTATGACGATCCCAGCAGT-3’20~25 bpswi6-W104A
¦¡xgRNA
20~25 bp

5’-GAGGCTATGAATACCTTTTGAAAGCGGAAGGTTATGACGATCCCAGCAGT-3’
TG →GC

PAMf3 bp6w/gn}b/9f0x2025 bpxVt >,q¡NMmc3W¡|ny@KW¡8ln¦=dt^d-;g)c3W¡s~-gHca

3. 2. N2zx¦DNAtlr3W¡=dy9 >$t4{r-\'gy@KW¡8ln¦=dt^d-;g)nclnc3W|n`_250~bpx@Vx ¦,qDNA/:¦DNAtlr$lr~^d\'s-kc
nolDNA/:PCRs%my@VxYw+ ¦xETlV%mxgZldc``_GFPvux4R?XdU"-mb25 bpx@V=lr~-g)gb
PCRsXd@Vx ¦,qDNA/:%lb¦DNAtlr$mjt~shc3. 2. N:±St²
. stn12Flag
stn1+
genomeZ!A25 bp ATGl\lZ!a­/|m£¯r5’\25bk

2 x Flag

25 bp

5’-agtctttaagggaatgacctaatATGGACTACAAGGACGACGATGACAAGGATTACAAAGATGACGACGATAAG(ATG)CTAGAAAATGAAAATCAACTA-3’

first ATG

25 bp

4. DNA

2 §¬¨£No5@³§¬¨£No5@fo% Ks*Gsnl N|RU<s{nr{Cas9 PL§¬¨£1}RU¢¡zquxlo)XY
2NoxstmCas9PL§¬¨£)XY2|t ¨¯D NzlPLs0yJ-RU¢¡|Cas9I"¥©4[vxstm)XY2|e ¨¯ z& u18e|m

1 §¬¨£No5@^?RUez ¨¯ zEMM5SB& u8e|m)XY2±]_­ ©@²

. S.pombe transformation kit (Wako)$3>9(1~2 x 10^7 cells/ml)RUcVzl1~2 x 10^9 cells/ml p.F|m´10 ml eB±1~2 x 10^7 cells/ml²cVz 100~200 ul  p=|mgRNAgRNA-Cas9PL§¬¨£(~1 ug)«°¦C~£¤°DNA ig;`­ig100 uM 10 ul (1 nmol)h;`­ig100 uM 4.5 ul x 2 : 900 pmoj
PCR4Hig300~ ngl4Htyª­DNA (2 mg/ml salmon testes DNA 2 ul)
1~2 x 10^7RU (10 ul)
Transformation reagent (45 ul)

37˚C2~3 7bdT|mz^?<sfE,+<W#E(M|± ´25˚C67br8²mRUyzu§®°¢'wm§®°¢32˚C6~76be|mzO%
W#E(e|± ´25˚C106bhm

RUsE(,+Q|l5. Knock-in5.1.

FG%vHz'¦j` 2v5`'+ft FG%wHxt`is)|jt Hz^YES u e!~au l_32˚Cr2^3(Ml_£v5`'+)|jt Hz^ EMMH# Otk¢uyh_32˚Cr¤^¥(Mk^3bq
fn:<YES u e!~au l_32˚Cr2^3(Ml_5.2. A1v7E
6u~oq:<eA18l^6 r7El uC&v
G%A1rH liserf_F

5.3. =?v7E
 YESsEMMH#uh^v=?7El_ura4+ 5`nw5-FOA rMkq?sliserf_5.4.  PCR=?kn Hz^ PCRcita

10 x PCR Buffer
2 mM dNTPs
primers (2.5 uM)

1 ul
0.8 ul
0.4 ul each
0.05 ul

Fw & Rv

Taq polymerase (S[]NRXWYZ\[WPO5 U/ul)
dH20

X ul

*yeast cells/10 ul
Q1~2(Mkn:<200 ulvvrppf^PCR".u-kqd vr:<>g/mq".u b_94˚C 2 min – (94˚C 30 sec. – 52 ˚C 40 sec.–72˚C (1 kb/ 1 min)) x 36 cycles – 72˚C 10 min– hold 12 ˚C

5.5. PCR 40vL*,
1 ulvPCR40 rL*,k^PCR40vfj¡6t;%b}I$¢sJ7El_5.6.   "u5`UTV40w9B{nw2vD@5`q  } v
dNTPKg_PCR product
ExoSAP-ITexpress

2.5 ul0.25 uldH20

0.75 ul/ 1 sample

37˚C 4 min – 80˚C 1 min – hold 4˚C
ExoSAPr 2kn1 ul v PCR 40 "u5l