Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

Abstract

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and Cas9-only control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 22% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Severe perinatal hydrops fetalis in genome edited pigs with a biallelic five base pair deletion of the Marfan syndrome gene, FBN1

“…We present a case of an unanticipated phenotype associated with CRISPR-Cas9 gene editing. We have previously reported the reinsertion of a deleted fragment close to the CRISPR target site in a mouse line (Summers et al, 2019b) and others have presented similar outcomes in edited human embryos, including loss of whole chromosomes (Alanis-Lobato et al, 2020; Liang et al, 2020; Zuccaro et al, 2020). It is not clear whether the phenotype in these piglets was due to an on-target event similar to these or an off-target event (although this was predicted to have a low probability by the guide design software), perhaps affecting the nearby GLDN gene, as we did not subject the DNA to whole genome sequencing.…”

“…Hydrops was recently seen in an infant with biallelic mutations in the gliomedin gene, GLDN , located 3 megabases downstream of FBN1 (Australian Genomics Health Alliance Acute Care Flagship, 2020; see Table 1 ). There have been a number of reports of DNA changes in the vicinity of a target sequence associated with the use of CRISPR-Cas9 gene editing (Summers et al, 2019; Alanis-Lobato et al, 2020; Liang et al, 2020; Zuccaro et al, 2020). It is possible that GLDN was mutated by the CRISPR-Cas9 process and is responsible for the hydrops fetalis phenotype in our piglets.…”

Human Germline and Heritable Genome Editing: The Global Policy Landscape

“…A clear and accurate understanding of the global policy landscape for heritable human genome editing is especially important in light of several recent developments, including the He Jiankui debacle 33 ; reports of other researchers stating that they intend to conduct clinical experiments aimed at producing genomeedited children 34 ; ongoing debate about the need for a global moratorium 32,[35][36][37] ; growing public and policymaker interest in, and concerns about, such experiments 38,39 ; recently published policy considerations on human genome editing 36,[40][41][42] ; advances in genome editing in human embryos, which confirm significant safety concerns [43][44][45][46] ; the recent report Heritable Human 47 To promote clarity and accuracy, this survey provides an overview of the existing global policy landscape for both heritable genome editing and germline genome editing-two related research practices with differing aims and consequences. Heritable genome editing involves the transfer of genetically modified embryos to a uterus to initiate a pregnancy that would result in the birth of a child with a modified genome.…”

Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option?

“…As a precaution, Alanis-Lobato et al [ 65 ] from the Francis Crick Institute have just reported a computational pipeline for single-cell genomics and transcriptomics of OCT4 ( POU5F1 ) CRISPR/Cas9-targeted and Cas9-only control human preimplantation embryos that allowed them to evaluate hard-to-detect on-target mutations. They observed loss-of-heterozygosity, segmental loss and gain of chromosomes, as well as unintended genome editing in analysed human embryo cells.…”