A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

Abstract

The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

“…Gene editing is deactivated by the anti-CRISPR during the G1 and S phases, and during the G2 phase, because the AcrIIA-CDT1 fusion is degraded, Cas9 becomes active. This provided not only a decrease of the off-target effects but, at the same time, a fourfold increase in HDR efficiency (Matsumoto et al, 2020). The efficiency was higher than in previous studies as the Cas9 is only inactivated but not degraded during the G1 phase.…”

CRISPR/Cas9-mediated Base-editing Enables a Chain Reaction Through Sequential Repair of sgRNA Scaffold Mutations

“…For example, the chain reaction system combined with the DNA barcoding technique will enable sequential cellular barcoding to understand individual cell fates with DNA memory. It will also be interesting to combine the cell cycledependent CRISPR-Cas9 activation system 12 with our chain reaction system to develop the counter of cell division. With such technology, we will be able to determine how many times the cell has divided from stem cells in future studies.…”

CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations

“…Thus, our chain reaction system provides a useful platform for recording and controlling molecular events in living cells. For example, in combination with the cell cycle-dependent CRISPR-Cas9 activation system 12 , our chain reaction system can be used to develop a counter for cell division. Previously, several methods to monitor cell division have been reported.…”