Multiplex CRISPR/Cas9 Assembly System Kit protocol

Multiplex CRISPR/Cas9 Assembly System Kit protocol (Yamamoto lab)
Ver. 1.1
July 2014
Tetsushi Sakuma, Ph. D.
E-mail: [email protected]
Takashi Yamamoto Lab.
Department of Mathematical and Life Sciences
Graduate School of Science
Hiroshima University
1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526 Hiroshima, Japan
TEL: +81-82-424-7446
E-mail: [email protected]
This is a protocol for the construction of all-in-one CRISPR/Cas9 vector expressing multiple (2-7)
gRNAs and a Cas9 nuclease or nickase described in Sakuma et al., Sci Rep, 2014
(http://dx.doi.org/10.1038/srep05400).
■Table of Contents
Materials
・・・・・・
2
1. Multiplex CRISPR/Cas9 assembly -STEP 1-
・・・・・・
3
2. Multiplex CRISPR/Cas9 assembly -STEP 2-
・・・・・・
4
・・・・・・
5
Methods
Appendix
1
■Materials
○Plasmids
Multiplex CRISPR/Cas9 Assembly System Kit (Addgene)
Insert plasmids:
pX330S-2, 3, 4, 5, 6, 7
(6 plasmids, Spectinomycin resistant)
Vector plasmids for Cas9 nuclease expression: pX330A-1x2, 1x3, 1x4, 1x5, 1x6, 1x7
(6 plasmids, Ampicillin resistant)
Vector plasmids for Cas9 nickase expression:
pX330A_D10A-1x2, 1x3, 1x4, 1x5, 1x6, 1x7
(6 plasmids, Ampicillin resistant)
○Reagents (excluding standard PCR enzymes and reagents for bacterial culture)
Maker
Product name
Cat. No.
Size/Unit
NEB
Quick Ligation Kit
M2200S
30 reactions
M2200L
150 reactions
ER0291
1000 units
ER0292
5000 units
Thermo Scientific
Eco31I*
NEB
10×T4 DNA Ligase Reaction Buffer
B0202S
6 ml
Life Technologies
ChargeSwitch-Pro Plasmid Mini Kit
CS30250
250 reactions
*BsaI (NEB, R0535S or R0535L) and BsaI-HF (NEB, R3535S or R3535L) can also be used.
○Primers
CRISPR-step2-F: GCCTTTTGCTGGCCTTTTGCTC
CRISPR-step2-R: CGGGCCATTTACCGTAAGTTATGTAACG
2
■Methods
1. Multiplex CRISPR/Cas9 assembly -STEP 1Insert the annealed oligonucleotides for gRNA templates. Since our vectors are derivatives of the
Feng Zhang lab’s pX330 plasmid (Addgene, Plasmid 42230), oligonucleotides should be designed
according to their previous report (http://dx.doi.org/10.1038/nprot.2013.143). Using our system, you
can make CRISPR/Cas9 vectors coexpressing 2-7 gRNAs and Cas9 nuclease/nickase. It is necessary
that you choose appropriate combinations of vectors for the desired applications.
DOI: 10.1038/srep05400
To perform STEP1 reactions, you can conduct conventional BpiI (BbsI) digestion and ligation
procedures. Alternatively, you can also conduct Golden Gate assembly-like method (see Appendix
for detailed reaction conditions).
Transform 0.5-1 µl of the reaction product directly to chemical competent E. coli such as XL1-Blue
and streak the transformant on Spectinomycin/Ampicillin-LB plate as described in Appendix.
3
Culture O/N at 37°C.
Pick two clones for each sample and culture them with 100 ng/µl Spectinomycin/Ampicillin-LB
media. Purify the plasmids using Miniprep kit.
To check the insert, BpiI digestion should be performed. If the oligonucleotides are correctly
inserted, the plasmids are no longer cut by BpiI.
2. Multiplex CRISPR/Cas9 assembly -STEP 2Perform 2nd-step assembly using the plasmids constructed in the section 1, following protocols
described in Appendix.
Transform
the
reaction
product
to
XL1-Blue
and
streak
the
transformant
on
Ampicillin/X-gal/IPTG-LB plate. Culture O/N at 37°C.
Pick white colonies and screen for correctly assembled clones by colony PCR. You can use
CRISPR-step2-F and CRISPR-step2-R primers both for pX330A and pX330A_D10A vectors. The
detailed reaction condition is described in Appendix.
DOI: 10.1038/srep05400
Culture the desired clones and purify the plasmids using Miniprep kit.
4
Multiplex CRISPR/Cas9 Assembly System Kit protocol
-AppendixBench protocol for STEP1 & 2 (Yamamoto lab)
STEP1
■Golden Gate reaction
(µl)
25 ng/µl vector
0.3
10 µM annealed oligos
0.5
10×T4 DNA ligase buf.
0.2
BpiI
0.1
Quick ligase
0.1
SDW
0.8
Total
2
37℃
16℃
4℃
premix → 1.2 µl each
→ Additional digestion:
5min. 10min. ∞
add 10×G buf. 0.2 µl, BpiI 0.1 µl.
premix → 0.3 µl each 37℃, 1 hr / 80℃, 5min. / 4℃, ∞
×3
■Transformation
Product
0.5
XL1-Blue (homemade)
10
on ice >5min. → 42℃, 30 sec. → on ice → plating
(100 µg/ml Spec or Amp plate)
5
STEP2
Number of gRNA cassettes
■Golden Gate reaction
2
100 ng/µl pX330S vector 1.5
3
4
5
6
7
1.5×2
1.5×3
1.5×4
1.5×5
1.5×6
50 ng/µl pX330A vector 1.5
10×T4 DNA ligase buf.
2
Eco31I
1
Quick ligase
1
SDW
13
Total
20
premix → 4 µl each
37℃
16℃
4℃
5min. 10min. ∞
11.5
10
8.5
7
5.5
×25
→ Additional digestion: add 10×G buf. 2 µl, Eco31I 1 µl.
premix → 3 µl each
37℃, 30min. / 80℃, 5min. / 4℃, ∞
■Transformation
Product
2
XL1-Blue (homemade)
20
on ice >5min. → 42℃, 30 sec. → on ice → plating
(100 µg/ml Amp plate w/ X-gal+IPTG)
■Colony PCR
10X buf. for Hybripol
0.8
dNTPs
0.64
10 µM primer
0.16+0.16 (CRISPR-step2-F/R)
Hybripol DNA polymerase (BIOLINE)*
0.04
MgCl2
0.24
95℃
95℃
62℃
72℃
72℃
4℃
SDW
5.96
30s.
15s.
15s.
90s.
50s.
∞
Total
8
×27
*Any other standard Taq polymerase can be used.
6

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