A group of researchers using a single method has promised to solve the problem of CRISPR-Case 9 with unwanted genetic mutations that allow it to stop gene editing until it reaches the stages of the main cell cycle where More repairs are likely.
Researchers at Hiroshima University and Tokyo Medical and Dental University published the results of their study on reduced study necrosis biology, which successfully demonstrated a more precise gene editing and called unwanted genetic removal, admission, or mutation a suppressed-target effect. Although previous methods were developed that reported low-target effects associated with CRISPR technology, the researchers said they often demonstrated low editing efficiency.
Our goal was to develop a so-called off-target effect which is one of the most challenging problems in the field of genome editing. “
Vataru Numura, one of the authors of the study, Professor, HU Graduate School of Biomedical and Health Sciences
“Our method is like killing two birds with one stone. We can improve the accuracy of genome editing and suppressing off-target effects at the same time.”
More control over gene-editing
CRISPR-Case 9 has launched a new front in gene editing as a simpler and less expensive tool. Acting like a scissor can deprive you of the genetic material you want to replace. The procedure, however, can also produce off-target effects that limit its use in the treatment field.
A new method developed to counteract the off-target effect works using the anti-CRISPR protein ACRII4, which acts as an “off switch” inhibiting the genome editing activity of Spikecase 9. Researchers have linked ACRII4 to the N-terminal region of human CDT1 – a gene that helps ensure that DNA replication occurs only once per cell division – with the intention of disabling gene editing from S and G2, The phase of the cell cycle when homeopathy-directed repair (HDR) is predominant.
HDR is one of the two DNA repair processes used by organisms with non-homogeneous end inclusion (NHEJ). Of the two, however, HDR is the preferred method because the repair depends on the presence of two chromosome copies in each cell. The use of duplicate chromosomes as a model for HDR repair makes gene editing more accurate as opposed to NHEG which only attaches to the broken end of DNA. Occurs during HDRS and G.2 Stages of the cell cycle while NHEJ works in all stages, especially in G.1 – The first stage of the interphase phase of the cycle where cell DNA replication progresses.
The researchers found that the amount of ARIIA 4-CDT1 fusion depends on the cell cycle. It grows during G.1 Stage that prevents gene-editing from occurring and, consequently, repairs by NHEJ. Meanwhile, it decreases during SG2, And M stages which are later.
“HDR efficiency using ACRII 4-CDT1 was increased approximately 4.0-fold compared to using Spikes 9 alone. At target or off-target site 1 (HCN1 genes), the conversion ratio was 86.5. In addition, the conversion ratio has dropped from 8.5% to 0.6% using the off-target site 2 (MFAP1 gene) ACIIA 4-CDT1, the researchers said in the study.
“The coexistence of Spicas 9 and ACRII 4-CDT1 not only increases HDR frequency but also suppresses off-target effects. Thus, the combination of Spicas 9 and ACRII 4-CDT1 is accurate and Cell cycle-dependent Case 9 activation system for efficient genome editing. “
Nomura said he wanted to improve the fairness of the system so that it could be used safely in the treatment field. “We envisage implementing our system for other CRISPR / anti-CRISPR combinations as well as other CRISPR based gene editors such as base editors and target transcription intermediaries.” “Our ultimate goal is to develop a genome editing system that can be used safely in the field of medical treatment.”
Matsumoto, D., And the rest. (2020) A cell cycle-dependent CRISPR-Case 9 activation system based on the anti-CRISPR protein improves the accuracy of genome editing. Communication Biology. doi.org/10.1038/s42003-020-01340-2.