The CRISPR-Cas system, which occurs naturally in certain bacteria and archaea, is an extremely important genetic tool in biotechnology. A deeper understanding and further development of this technology will enable advances in numerous fields such as genetic engineering, disease treatment, agriculture, food industry, ecology, pharmacy and many others. The most commonly used system is based on the Cas9 endonuclease and its derivatives (dCas9, nCas9). There are several applications of the CRISPR-Cas9 system. Among the most important for genetic engineering are the classic CRISPR-Cas9 system and the modified systems that enable gene regulation, base editing and epigenetic modifications. A more recent approach is prime editing, which enables the insertion or deletion of small sequences at target sites in addition to editing of individual nucleotides. The latter represents a major breakthrough in this field, as base editors and prime editing systems have successfully overcome the limitations of classic CRISPR-Cas9. Despite its enormous potential, the technology faces several limitations, the most critical of which are off-target effects. As these are unacceptable in in vivo applications, it is imperative that they are eliminated. In addition to these limitations, there are also ethical concerns, particularly in relation to genome editing of embryos and germ cells, which could lead to the transmission of changes to future generations and thus to unpredictable outcomes.
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