Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Summary

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Abstract

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to β-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

Introduction

In 2011, researchers proposed a computational method and developed a corresponding piece of software for the automatic design of digital synthetic gene circuits¹. A user had to specify the number of inputs (three or four) and fill in the circuit truth table; this provided all the necessary information to derive the circuit structure using techniques from electronics. The truth table was translated into two Boolean formulae via the Karnaugh map method². Each Boolean formula is made of clauses that describe logic operations (sum or multiplication) among (part of) the circuit inputs and their negations (the literal....

Protocol

1. Design and construction of the sgRNA/crRNA expression cassette

NOTE: There are two kinds of sgRNA/crRNA expression cassettes: one-termed SNR52¹⁰-is composed of the RNA polymerase III-dependent SNR52 promoter, the sgRNA/crRNA sequence, and the SUP4 terminator; another-abbreviated as RGR¹¹-consists of the RNA polymerase II-dependent ADH1 promoter, the RGR (ribozyme-guide RNA-ribozyme) structure that contains two ribozymes (a hammerhead ribozyme-HH, and a hepatitis delta virus ribozyme-HDV) and the sequence of the sgRNA/crRNA in-between, and the AD....

Discussion

The protocol showed a possible complete workflow for synthetic gene digital circuits, following the "Design-Build-Test-Learn" (DBTL) biological engineering cycle and concerning both dry-lab and wet-lab experiments. Here, we focused on the CRISPR-Cas system, mainly dSpCas9, denAsCas12a, dLbCas12a, and the corresponding anti-CRISPR proteins, by designing and building in S. cerevisiae small transcriptional networks. Some of them mimicked Boolean gates, which are the basic components of digital circuits. All.......

Materials

Name	Company	Catalog Number	Comments
0.1 mL PCR 8-strip tubes	NEST	403112
0.2 mL PCR tubes	Axygen	PCR-02-C
1.5 mL Microtubes	Axygen	MCT-150-C
15 mL Centrifuge tubes	BIOFIL	CFT011150
2 mL Microtubes	Axygen	MCT-200-C
50 mL Centrifuge tubes	BIOFIL	CFT011500
Agarose-molecular biology grade	Invitrogen	75510-019
Ampicillin sodium salt	Solarbio	69-52-3
Applied biosystems veriti 96-well thermal cycler	Thermo Fisher Scientific	4375786
AxyPrep DNA gel extraction kit	Axygen	AP-GX-250
BD FACSuite CS&T research beads	BD	650621	Fluorescent beads
BD FACSVerse flow cytometer	BD	-
Centrifuge	Eppendorf	5424
Centrifuge Sorvall ST 16R	Thermo Fisher Scientific	75004380
E. coli competent cells (Strain DH5α)	Life Technologies	18263-012
ECL select Western Blotting detection reagent	GE Healthcare	RPN2235
Electrophoresis apparatus	Beijing JUNYI Electrophoresis Co., Ltd	JY300C
Flat 8-strip caps	NEST	406012
Gene synthesis company	Azenta Life Sciences		https://web.azenta.com/zh-cn/azenta-life-sciences
Goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 568	Invitrogen	A-11004
HiFiScript cDNA synthesis kit	CWBIO	CW2569M	Kit used in step 6.2.2.1
Lysate solution (Zymolyase)	zymoresearch	E1004-A
Nikon Eclipse 80i fluorescence microscope	Nikon	-	Fluorescence microscope
Pipet tips—10 μL	Axygen	T-300-R-S
Pipet tips—1000 μL	Axygen	T-1000-B-R-S
Pipet tips—200 μL	Axygen	T-200-Y-R-S
pRSII403	Addgene	35436
pRSII404	Addgene	35438
pRSII405	Addgene	35440
pRSII406	Addgene	35442
pRSII424	Addgene	35466
pTPGI_dSpCas9_VP64	Addgene	49013
Q5 High-fidelity DNApolymerase	New England Biolabs	M0491
Restriction enzyme-Acc65I	New England Biolabs	R0599
Restriction enzyme-BamHI	New England Biolabs	R0136
Restriction enzyme-SacI-HF	New England Biolabs	R3156
Restriction enzyme-XhoI	New England Biolabs	R0146
Roche LightCycler 96	Roche	-	Real-Time PCR Instrument
S. cerevisiae CEN.PK2-1C	-	-	The parent strain. The genotype is: MATa; his3D1; leu2-3_112; ura3-52; trp1-289; MAL2-8c; SUC2
Stem-Loop Kit	SparkJade	AG0502	Kit used in step 6.2.1.3
T100 Thermal Cycler	BIO-RAD	186-1096
T4 DNA ligase	New England Biolabs	M0202
T5 Exonuclease	New England Biolabs	M0363
Taq DNA ligase	New England Biolabs	M0208
Taq DNA polymerase	New England Biolabs	M0495
TB Green Premix Ex Taq II (Tli RNaseH Plus)(2x) (SYBR Green I dye)	Takara	RR820Q
YeaStar RNA kit	Zymo Research	R1002
β-estradiol	Sigma-Aldrich	E8875