CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.

CRISPR–Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR–Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR–Cas-assisted phage engineering, beyond single pathogen control.

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This research was conducted under the auspices of VLAG Graduate School (Biobased, Biomolecular, Chemical, Food, and Nutrition sciences) History of genetic engineering Artificial selection and molecular biologyExploring the infinite complexity of nature, humankind has always been seeking for new means to satisfy primary needs, such as food and health (Fig. 1). In prehistoric years, hunting and gathering of wild animals and plants were the predominant modes of subsistence, being steadily replaced by domestication and selective breeding in early societies 1,2 . The term 'selective breeding' was first coined by Robert Bakewell during the British Agricultural Revolution (1783) 3 , spurring the conceptualization of natural selection (by Charles Darwin, 1859) 4 , genetic inheritance (by Gregor Mendel, 1865) 5 , (pan)gene as the unit of hereditary information (by Hugo de Vries, 1889) 6 , and genetics (by William Bateson, 1905) 7 . Although the study and the development of artificial selection techniques facilitated screening of animals and plants for desired traits, direct intervention in the genetic variation was unattainable. In the late 1920s, Hermann Joseph Muller subjected flies to high temperature or X−ray radiation to deliberately introduce random genetic mutations, which were inherited to the next generation 8-10 . In the same period, Frederick Griffith's experiment showed that genetic information can be naturally transmitted among bacteria via a process called transformation 11 . This genetic information was later identified as deoxyribonucleic acid (DNA) (by Oswald Avery, Colin MacLeod and Maclyn McCarty) 12 and its double−helix structure was resolved in 1953 (by James Watson, Francis Crick, Maurice Wilkins, and Rosalind Franklin) 13 . In 1958, the Central Dogma of molecular biology revealed that genetic information passes from DNA (in the form of a gene) to RNA and then to protein, allowing for the genetic information stored in genes to be expressed as proteins with a wide spectrum of catalytic and structural functions 14 . In the period 1950−1970, a series of seminal discoveries (plasmid DNA, DNA ligases, restriction enzymes) and the invention of artificial (chemical) transformation led to creation of the first recombinant DNA (Berg, 1972) [15][16][17][18][19] . Recombinant DNA technology and genome editingThe generation of the first genetically modified (GM) bacteria, animals, and plants symbolized the dawn of the genetic engineering era [20][21][22] (Fig. 1). In 1977, a human protein (somatostatin) produced by GM Escherichia coli was commercialized for the first time.