Protocols for C-Brick DNA Standard Assembly Using Cpf1

Summary

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

Abstract

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites – the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) – flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short "GGATCC" scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

Introduction

The standardization of DNA biological parts is important for the development of synthetic biology¹. The development of a DNA assembly procedure can replace ad hoc experimental designs and remove many of the unexpected outcomes that arise during the assembly of genetic components into larger systems. The BioBrick standard (BBF RFC 10) was one of the earliest proposed DNA assembly standards. It uses the prefix sequence (containing EcoRI and XbaI cutting sites) and the suffix sequence (containing SpeI and PstI cutting sites)^2,3. Because XbaI and SpeI have complementary cohesive en....

Protocol

1. Preparation of crRNA

1. Preparation of crRNA templates
   1. Re-suspend individual oligonucleotides (Table 1) in RNase-free water to a concentration of 10 µM.
   2. Add 22.5 µL of top-strand oligonucleotide (T7-F in Table 1), 22.5 µL of bottom-strand oligonucleotide (Table 1), and 5 µL of 10x annealing buffer to a 0.2 mL PCR tube. Ensure that the total volume is 50 µL.
      NOTE: Six different bottom-strand oligonucleotides are shown in Table 1, each of which should be individually paired with the top-strand T7-F. The lowercase letter....

Results

This protocol demonstrated the assembly of three chromoprotein cassettes (cjBlue (BBa_K592011), eforRed (BBa_K592012), and amilGFP (BBa_K592010)). First, the coding sequences of the three abovementioned genes and terminators were individually cloned into a C-Brick standard vector. Short DNA parts, promoter and terminator, were introduced into the C-Brick vector through PCR amplification using primers containing the short DNA parts on the 5' terminal. This was followed by the self-ligation.......

Discussion

This protocol describes a procedure for the DNA assembly standard C-Brick. The most important step in this protocol is the linearization of the C-Brick standard vector; incomplete cleavage of the vector could seriously affect the success rate. Additionally, although Cpf1 mainly cleaves target DNA sequences in the "18-23" cleavage pattern, inaccurate cleavage near the two bases was also detected⁴, which can cause a small number of mutations after DNA assembly. Therefore, Sanger sequencing i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Comercial Oligonucleotide	Sangon Biotech
10x Taq PCR Buffer	Transgen	#J40928
Ultra Pure Distilled Water	Invitrogen	10977-015
5x RNA Transcription Buffer	Thermo Scientific	K0441
T7 RNA polymerase	Thermo Scientific	#EP0111
NTP mixture	Sangon	#ND0056
RRI (Recombinant RNase Inhibitor)	Takara	2313A
RNA Clean & Concentrator-5	Zymo Research	R1015
UV-Vis Spectrometer	Thermo Scientific	Nano-Drop 2000c
2x Phanta Max Buffer	Vazyme	PB505	PCR buffer
dNTPs	Transgen	AD101
Phanta Max Super-Fidelity DNA Polymerase	Vazyme	P505-d1
Ezmax for One-step Cloning	Tolobio	24303-1	seamless assembly kit
5x Buffer for Ezmax One-step Cloning	Tolobio	32006
BamHI	NEB	#R0136L
BamHI-HF	NEB	#R3136L
BglII	NEB	#R0144L
XbaI	NEB	#R0145L
SpeI	NEB	#R3133L
10x Buffer 3	NEB	#B7003S
10x CutSmart Buffer	NEB	B7204S
10x T4 DNA ligase Buffer	Tolobio	32002
T4 PNK	Tolobio	32206
T4 DNA ligase	Tolobio	32210
DpnI	NEB	#R01762
SV Gel and PCR clean-up system	Promega	A9282
Plasmid Mini Kit I	Omega	D6943-02	plasmid preparation kit
thermosensitive alkaline phosphatase	Thermo Scientific	#EF0651	FastAP
10x Cpf1 buffer	Tolobio	32008
Cpf1	Tolobio	32105	FnCpf1
thermocycler	Applied Biosystems	veriti 96 well
C-Brick standard vector	Tolobio	98101
E. coli [DH10B]	Invitrogen	18297010
Luria-Bertani media (tryptone)	Oxoid	LP0042
Luria-Bertani media (yeast extract)	Oxoid	LP0021
Luria-Bertani media (NaCl)	Sangon Biotech	B126BA0007