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When developing engineered bacteria using plasmid expression systems, the introduction of plasmids
carrying exogenous DNA into host bacteria typically involves screening for successful transformation using
antibiotic resistance genes. However, the inclusion of these resistance genes in engineered bacteria poses a
barrier to their clinical application. To address this issue, researchers are exploring alternative selection
markers such as sugar utilization genes, phage resistance factors, and stress tolerance mechanisms.
Additionally, emerging gene editing technologies like the CRISPR-Cas system offer a promising avenue for
genome editing in bacteria, enabling the creation of antibiotic-free engineered bacteria . Currently, most
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synthetic biology tools are primarily developed for EcN and Lactobacillus, with relatively few engineering
tools available for strict anaerobic bacteria found in the colon . Additionally, the regulatory framework for
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engineered bacteria as novel therapeutics remains incomplete, and regulatory requirements may vary across
countries and regions, presenting a challenge for global clinical trials and product launches.

SYNTHETIC BIOLOGY AND CRISPR/CAS SYSTEM
The synthesis of proteins and DNA, combined with the establishment of biocentric laws, laid the
foundation for the idea of creating living organisms from scratch, leading to the rapid development of
synthetic biology. Synthetic biology is an interdisciplinary field that integrates concepts and techniques
from biology, engineering, computer science, and molecular biology. It is typically defined as the redesign
or engineering of existing biological components and systems using specific technologies to achieve a
desired function . In 2010, Gibson et al. created the first synthetic genomic cell, employing a bottom-up
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approach in which chemically synthesized DNA fragments were assembled to form a minimal genome
necessary for the basic functioning of an organism . This minimal genome offers advantages in stability
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and efficiency, but challenges remain, such as the difficulty of synthesizing long DNA sequences from
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scratch . In contrast, the field of engineered bacteria often adopts a top-down strategy, which involves
modifying existing organisms by removing non-essential genes from their genomes. This process frees up
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space and energy to optimize the expression of target genes . For example, studies have shown that
heterologous protein production in Lactobacillus lactis NZ9000 is significantly improved when 2.83% of the
non-essential genome is deleted using the Cre-loxP deletion system . Plasmid-based expression systems
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are commonly used in engineered bacteria, but they face limitations for clinical applications due to issues
with genetic instability and the potential for biological contamination. As a result, gene-editing
technologies, particularly those based on the CRISPR-Cas system, are emerging as powerful tools. These
technologies have the potential to stabilize and modify probiotic genomes, enabling the alteration of their
metabolic pathways and biological properties for more precise and reliable applications.

The CRISPR/Cas system is an acquired immune mechanism found in most archaea and approximately half
of all bacteria, where it functions to recognize and excise invading viral DNA . This system consists of two
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main components: the sequence responsible for encoding Cas-related proteins and the CRISPR array, which
contains a series of repeats interspersed with spacer sequences. The spacer sequences store fragments of
foreign genetic material, allowing the organism to “remember” previous invaders . The mechanism
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operates through three main stages: Adaptation: The complex formed by the Cas1 and Cas2 proteins
recognizes foreign genetic material and integrates it into the spacer sequences of the CRISPR array.
Expression: The CRISPR array is transcribed into pre-crRNA, which is then processed into mature crRNA
by Cas proteins. Interference: The crRNA binds to the Cas protein to form a complex that identifies and
cleaves foreign DNA sequences that are complementary to the spacer sequence . This recognition occurs
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only when the crRNA binds to the protospacer adjacent motif (PAM) sequence, a short DNA motif located
adjacent to the target sequence . Based on this mechanism, the CRISPR/Cas system has been developed
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into a powerful tool for gene editing. Unlike conventional technologies such as zinc finger nucleases (ZFNs)
and transcription activator-like effector nucleases (TALENs), which rely on protein-DNA recognition,