Speaker- Raymond H.J. Staals

Clustered, regularly interspaced short palindromic repeats (CRISPR-Cas), a groundbreaking bacterial immune system adapted for genetic editing, was declared the scientific breakthrough of the year in 2015 and has garnered widespread media attention. It has sparked debates and featured prominently in television programs. The technology has been widely embraced, culminating in the Nobel Prize in Chemistry awarded to Jennifer Doudna and Emmanuelle Charpentier in 2020. CRISPR-Cas is ubiquitous across scientific disciplines, influencing discussions and appearing frequently in scientific presentations and publications. Its rapid adoption underscores its transformative impact in various fields. The increasing volume of CRISPR-related study since 2008 reflects ongoing discoveries of novel CRISPR-Cas systems, dispelling misconceptions, elucidating natural roles, and evolving into sophisticated genetic tools.

The impact of viruses on society, exemplified by the COVID-19 pandemic, underscores their significant influence on human health and global systems. Bacteria face an immense viral threat, with an estimated 1024 viruses attempting to infect each bacterium. Bacteria combat viral infections by employing unique defense mechanisms such as CRISPR-Cas systems. Viruses inject their Deoxyribonucleic acid (DNA) into bacterial cells in the initial stages of disease, hijacking cellular machinery to produce new viral particles. Upon completion of the viral life cycle, the bacterial cell ruptures, releasing progeny viruses capable of infecting other cells. CRISPR-Cas is a critical defense mechanism in bacteria, providing acquired immunity by storing viral DNA sequences and using them to recognize and cleave viral DNA during subsequent infections.

CRISPR-Cas immunity involves three stages: adaptation, expression, and interference. During adaptation, fragments of viral DNA or Ribonucleic Acid (RNA) are integrated into the bacterial CRISPR array. In the expression stage, the CRISPR array is transcribed into CRISPR RNA (crRNA), each crRNA containing a specific sequence from a sampled virus. The expression of Cas proteins, including proteins like Cas9, follows. In the interference stage, Cas proteins utilize the crRNA as a guide to locate and cleave viral DNA or RNA, functioning akin to a surveillance and defense system against viral threats.

The CRISPR-Cas system was discovered in 2007 following experiments involving bacteria and viruses. Studies observed that when bacteria were equipped with CRISPR-Cas systems containing specific spacer sequences, they became immune to viral infections by targeting and cleaving viral DNA or RNA. The breakthrough led to the system's initial application in genome editing. CRISPR-Cas technology could enhance productivity in the dairy industry by engineering production strains resistant to viral infections.

CRISPR-Cas systems, such as Cas9 and Cas12, are specialized nuclease enzymes categorized into classes 1 and 2. Class 1 systems utilize multiple genes and proteins, while Class 2 systems typically involve a single large protein for interference, adaptation, and expression of crRNA. In Class 1 systems, crRNA guides Cas proteins to target and cleave viral DNA or RNA. The most significant breakthrough in genome editing was the development of customizable guide RNAs, enabling Cas9 proteins to cleave targeted DNA sequences, including viral genomes, precisely.

CRISPR-Cas systems are primarily adaptive immune systems in bacteria and archaea, but in genome editing applications, Cas proteins like Cas9 and Cas12 can induce targeted DNA double-strand breaks (DSBs). These breaks can be repaired by two main pathways: non-homologous end joining (NHEJ), which directly ligates broken DNA ends but can be error-prone, and homology-directed repair (HDR), which uses homologous DNA sequences to repair DSBs with high fidelity. CRISPR-Cas technology enables precise genetic modifications by facilitating gene knockout, insertion, or correction, depending on cellular conditions or tissue.

Over the past decade, CRISPR-Cas9 technology has profoundly impacted human genome editing, embryology, and agriculture, developing larger crops and novel traits. While CRISPR-Cas9 has enhanced agricultural productivity, regulations in regions like the European Union aim to balance innovation with safety concerns to avoid competitive disadvantages. CRISPR-Cas9 has also advanced into clinical trials in the medical sector, with the first patient treated in the Netherlands two years ago.

CRISPR-Cas systems, consisting of CRISPR sequences and Cas proteins like Cas9, are versatile tools with numerous applications. While CRISPR-Cas9 is primarily used for genome editing, CRISPR technologies extend to epigenetics, diagnostics, and forensics. They can modulate gene expression indirectly through epigenetic modifications or be adapted for diagnostic tests targeting specific DNA or RNA sequences, such as those from viruses like coronavirus or Zika. CRISPR-Cas systems can also record biological processes within bacterial DNA in real time, illustrating their revolutionary impact across various scientific fields.

Cas9 nuclease has made significant strides in genome editing by recognizing specific DNA sequences guided by complementary RNA sequences. However, off-target effects remain a concern in human experiments. Biophysical approaches are used to study Cas9's interactions with DNA, although labeling with dyes like purple dye is not a standard method for programming Cas9. Delivering Cas9 into cells can be costly and may require techniques such as microinjections. Viral vectors have been explored to deliver Cas9 into cells throughout the body for potential therapeutic applications in animals and humans. Still, challenges such as immune responses and off-target effects persist.

The ethical implications of genetic engineering, particularly in newborns, include concerns over unintended consequences such as genetically modified mosquitoes carrying disease-causing genes, potentially leading to increased mosquito populations. Specific techniques like EGPRIME (Engineered Genetic Pest Reduction with Inheritable Modification), raise ethical questions regarding safety and societal impact. Gene drives, which spread desired genes through populations, suggest collaborative efforts between humans and technology to manage gene inheritance, though skepticism about their control persists. Discussions around CRISPR-Cas, initially an adaptive immune system in bacteria, explore potential benefits like enhancing immunity to diseases such as coronavirus, raising ethical considerations about germline editing and inherited traits. Addressing these ethical, technical, and legislative challenges requires ongoing study and careful regulation.

European Society of Human Reproduction and Embryology, July 7-10, Amsterdam, The Netherland