Researchers at the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch and the Berlin Institute of Health (BIH) have engineered a more efficient CRISPR-Cas9 genome editing tool. The authors of the Nature Biotechnology article inhibited key molecules of the Non-Homologous End Joining (NHEJ) repair pathway, thus boosting the more precise Homology Directed Repair (HDR) pathway. This innovation increased the efficiency of the CRISPR-Cas9 technology more than eightfold.
Genome editing tools have been sought after for many years in biomedical research. Several technologies have been developed in the past, such as RNA interference (RNAi) or transcription-activator like effector nucleases (TALENs). These technologies, although welcome, had several drawbacks: depending on the case, they were expensive, slow, imprecise or transiently effective. The discovery of the function of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated Cas genes –a bacterial defense system against extraneous genetic material- prompted the Doudna group from the Howard Hughes Medical Institute – University of California at Berkeley to use it as a new tool to edit genes. In some bacteria and archaea, foreign DNA is recognized and incorporated at the CRISPR loci; after its transcription, RNA is processed into crRNA, which binds Cas endonucleases and targets them to cleave alien, complementary DNA sequences. The Doudna lab recognized its potential and engineered the crRNA sequence to match that of any desired locus in the genome, which would then be targeted by Cas9 and nicked. Double strand breaks (DSBs) would then be repaired either by NHEJ or HDR. NHEJ is more efficient but less precise that HDR, as it reattaches the DNA without any template. This is convenient for generating random mutations to silence a gene, but not for inserting a specific sequence. HDR is less efficient but can be used to generate precise mutations by inserting a donor DNA whose flanking regions are complementary to the targeted locus.
Mammalian cells rely mostly on NHEJ for DSB repair, but Van Trung Chu et al. found a way to enhance the HDR pathway. They temporarily inhibited the NHEJ key molecules KU70, KU80 and DNA Ligase IV with small molecules and adenovirus proteins. These molecular modifications improved HDR efficiency up to eightfold and almost abolished NHEJ in human and mouse cell lines.
The CRISPR-Cas technology is only 3 years old and is currently one of the most interesting tools for biologists, not only because of what is has achieved so far, but also because of the possible future innovations and modifications.