# Phase Therapy: Consider the Past, Embrace the Future
## 3 Strategies for Phage Therapy
### Phage Combined with CRISPR-Cas System  
Strictly speaking, the combination of a phage and CRISPR-Cas system is also a kind of phage engineering, but we list it separately as a section here, mainly because of its novelty and importance. The clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) system in bacteria and archaea provides sequence-based adaptive immunity against mobile genetic elements such as viruses and plasmids [82]. In general, the CRISPR-Cas system acts by integrating the short fragments of nucleic acid of viruses or plasmids into the CRISPR array and produce short RNA sequences complementary to them (known as CRISPR RNAs, namely crRNAs), and these resultant crRNAs guide Cas protein complex to specifically target invading foreign genetic elements for degradation [83]. Based on the precise sites interference of CRISPR-Cas and the high infection efficiency of a phage, we can implement the delivery of the CRISPR-Cas system with phage to specifically target bacterial genomic DNA (or RNA) so as to eliminate pathogenic bacteria and prevent the emergence of bacterial resistance [84]. For the target bacteria containing an active endogenous CRISPR-Cas system, we only need to embed a mini-CRISPR array into the phage genome so as to express the artificial crRNA matching drug-resistant bacterial genome. In short, the endogenous CRISPR-Cas system is exploited by bacteriophage to attack its host cell [85]. More recently, after a phage encodes bacterial genome-targeting CRISPR array, the endogenous CRISPR-Cas3 in _C. difficile_ is repurposed as an antimicrobial, resulting in the bacterial cells death through the phage infection lysis or CRISPR effector complexes self-targeting [51]. For the pathogens without a CRISPR-Cas system, an additional, exogenous CRISPR nuclease could be a good choice. In several previous studies, it has been reported that the use of phagemids with exogenous CRISPR-Cas9 system results in the specific and rapid bacteria targeting [38], and that the programmed CRISPR-Cas13a is packed into a bacteriophage capsid to target antimicrobial resistance genes [52]. Similarly, _S. aureus_ phage \(\varphi\) NM1 is used to deliver an exogeneous CRISPR-Cas system to eliminate the pathogenic host [37]. A temperate phage-based delivery system is used to deliver CRISPR/Cas9 against bacteria [45]. Excitingly, some phages encode their own CRISPR-Cas systems, including types II and V, which are widely used for gene editing, and the related effector proteins have complete catalytic residues, indicating that these effector proteins are functionally active [86; 87; 88; 89]. At the same time, we should also note that some bacterial genomes encode anti-CRISPR (Acr) proteins which can inhibit the CRISPR-Cas system in many ways [90]. Another obvious advantage of the bacteriophage combined with the CRISPR-Cas system is that it allows bacteriophages to be programmed to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria, which is of great significance to avoiding the emergence of resistant bacteria [91]. To sum up, the phage combined with CRISPR-Cas system has a splendid application prospect in the prevention and treatment of drug-resistant bacteria.