Highly Efficient CRISPR/HDR-Mediated Knock-in for Mouse Embryonic Stem Cells And Zygotes

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system

“…First, the donor DNA requires only two short homology arms of < 100 nt and thus can be easily synthesized. This is in contrast with traditional CRISPR/Cas9-mediated precise editing which requires long homology arms of a few kbs 9,10,11,12 . Second, this system does not cause additional toxicity compared with the CRISPR/Cas9 system, as evidenced by a similar survival rate of the F0 pups in embryos manipulated by these two systems.…”

“…The precise knock-in of a larger DNA fragment, such as one as large as 1 000 bp, is still a challenging task in mice 8 . Even for a donor DNA with ∼1-8 kb homology arms, the knock-in efficiency achieved by CRISPR/Cas9 was lower than 6% in mice 9,10,11,12 .…”

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

“…Nevertheless, there are reports of DSD templates injected into the mouse zygote for the integration of reporter transgenes 70, 71 or a conditional allele. 72 These studies utilized conventional homology arms of variable lengths (3–9 kb) in a circular DSD template; linearization of the DSD donor is not done so as to reduce random integration in the mouse genome.…”

The potential and pitfalls of de‐extinction

“…Furthermore, currently the insertion of a new strand of DNA cannot be completely controlled, and occurs at a lower frequency than the simple repair of the break which the Cas9 produces, with reports in the range of 0.5–20% of cuts resulting in successful insertions (Wang et al . ). This is due to non‐homologous end joining being the primary mechanism used by cells to repair double‐strand breaks: a process which often results in random deletions and insertions during the repair process.…”

“…), these molecules may interfere with embryo development (Wang et al . ), and regardless, the efficiency is still fairly low, which may pose a problem for large scale genome editing. After each attempted deletion and insertion, checks would be required to see whether the insertion was actually made.…”