Genome-editing technology in CRISPR/Cas system: How to increase knock-in efficiency in mouse zygotes

Abstract

The use of clustered regularly interspaced short palindromic repeats (CRISPR) and RNA-guided Cas9 nucleases, known as the CRISPR/Cas system, represents a major technological advance in mammalian gene disruption. CRISPR/Cas enables genome editing by inducing targeted DNA double-strand breaks (DSBs) that are repaired by error-prone, nonhomologous end-joining (NHEJ), or homology-directed repair (HDR). This system has emerged as an effective tool for gene knockout via NHEJ; however, it remains ineffi cient for precise editing of genome sequences depending on HDR. Nevertheless, HDR-mediated gene editing is essential for conditional knockout, introduction of reporter genes, and precise point mutation in mice. Many studies have examined, for example, conditions of Cas9 and guide RNA (gRNA), methods of their introduction, and molecules to increase effi ciency. In this review, we describe various methods for increasing the effi ciency of editing in mouse zygotes.