As the potential application of genome editing grows, risks appear which must be rigorously analyzed.
Genome editing is the modification or removal of specific DNA sequences in order, for example, to correct a disease-causing mutation. First approaches were based on the recognition of specific sequences using oligonucleotides, small molecules or self-splicing introns. Later, techniques based on DNA sequence recognition by proteins were developed, such as site-directed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENS). These methods are based on binding of a DNA cleavage protein domain to a zinc finger or TALE DNA binding domain, respectively, which has been modified to target the desired DNA sequence. However, difficulties in the design, synthesis and validation of these proteins are an obstacle to the widespread adoption of these artificial nucleases.
One new genome editing technique is CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9, which is based on a system described in bacteria that gives these microbes adaptive immunity against viruses. In natural bacterial systems, some genomic sequences of viruses that infect bacteria are incorporated into the bacterial genome between CRISPR sequences. Thus, if the same virus attacks it again, the bacteria produces an immune response that includes a copy of the “remembered” sequences, called crRNA, which bind to virus DNA, and a second RNA, called tacrRNA, which recruits a Cas endonuclease to cut the virus DNA. The technique, patented by Jennifer Doudna and Emmanuelle Charpentier in 2012, consists of modifying the tracrRNA:crRNA pair as a single guide RNA (sgRNA) with a sequence at the 5′ end which determines the DNA target sequence and a duplex RNA structure at the 3′ end, which binds to Cas9 .
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