P-806 Heritable genome editing in haploid mouse embryonic stem cells
Authors: Pizzo E , De Jesus A , Xie P , Lari E , Zev R , Palermo G 
Published: July 2024

Abstract

Study question
Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells?

Summary answer
We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation.

What is known already
Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism.

Study design, size, duration
In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype.

Participants/materials, setting, methods
Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay.

Main results and the role of chance
A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%.

Limitations, reasons for caution
While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele.

Wider implications of the findings
The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny.

Trial registration number
Not applicable