Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications

Table 2 Escape frequencies of selected biosafety systems

Name of the System	Type of System	Escape Frequency	Reference
SLiDE, single allele	Auxotrophy	8 × 10^− 4 to 3 × 10^− 9	[69]
SLiDE, two alleles	Auxotrophy	5 × 10^− 10	[69]
SLiDE, three alleles	Auxotrophy	< 3 × 10^− 11	[69]
Thymine/Thymidine auxotrophy	Auxotrophy	Below detection limit	[71, 72]
Artificial Phosphite Dependency	Auxotrophy	1.94 × 10^− 13	[70]
Single ncAA auxotrophy	Auxotrophy/Xenobiology	No escape mutants in >5 × 10¹¹ cells	[381]
Triple ncAA auxotrophy	Auxotrophy/Xenobiology	6.41 × 10⁻¹¹	[57]
CcdB	Kill switch	~ 10^− 3	[28]
Cryodeath	Kill switch	< 1 in 10⁵ after 10 days in vivo	[107]
Deadman	Kill switch	Below detection limit	[28]
Passcode	Kill switch	Below detection limit	[28]
CRISPR mediated DNA degradation	DNA destruction	Viable cells reduced by a factor of 10⁸	[152]
Thermoinduced DNA degradation	DNA destruction	2 × 10^–5	[135]
GeneGuard	Combinatorial system	Below detection limit	[32]
SafeGuard	Combinatorial system	<1.3 × 10^− 12	[76]

In general, the combination of several systems reduces the probability for random mutagenesis to disarm the biosafety system and for cells to bypass the biosafety system. Therefore, multilayered systems like Passcode, Deadman or GeneGuard act as great examples for complex biosafety systems that achieved very low escape frequencies. Engineering artificial auxotrophies, such as an artificial phosphite dependency, can also act as potent biosafety systems, as shown by Hirota et al.

ISSN: 1754-1611