CRISPR gene editing faces a "pedigree conundrum": the genetic diversity of Africans leads to off-target effects

CRSIPR gene editing technology has been born for 10 years, in the past 10 years, CRISPR gene editing has achieved rapid development with the efforts of scientists around the world, and has become the most simple and efficient gene editing tool, and has made breakthroughs
in gene function research, drug target screening, genetic disease treatment, cancer research, crop breeding and other fields.

When using CRISPR technology for genome editing, a guide RNA (gRNA) needs to be designed to identify and target the target genomic loci
we need to edit.
Under gRNA guidance, Cas enzymes reach the correct genomic loci and cut and edit
DNA.
If the gRNA does not match the target genomic locus enough, the CRISPR system may not be able to complete the required editing
.

Today, a number of CRISPR-based gene therapies have been used in human clinical trials, however, laboratory mice are usually inbred and genetically identical, but the human genome diversity is high, depending on the bloodline
.

These differences in ancestry mean that CRISPR does not always edit some genomic loci as expected, especially in people of African ancestry, as their genomes may differ from
those used to design guide RNAs (gRNAs).

Recently, researchers from the Broad Institute in the United States published a research paper
entitled: Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries on the preprint platform bioRxiv.

The study shows that because the reference genome used in the gRNA design process for CRISPR gene editing often does not consider lineage, important cancer target genes
in people of African ancestry may be missed in cancer gene research.
The study also developed an online tool to help assess the impact
of ancestry problems on standard CRISPR-gRNA.

Wizard RNA (gRNA) is often designed based on sequencing compiled reference genomes from a small number of human genomes, which are not representative of the diversity
of the human genome.
For example, people of African descent are genetically more diverse than Europeans and Asians because their ancestors traveled out of the continent and spread around the world
in a relatively recent time.

In this study, the team used the Cancer Dependency Map to explore the "pedigree puzzle"
of CRISPR gene editing.
The Cancer Dependency Atlas is a large-scale research project in collaboration with the Wellcome Sanger Institute in the United Kingdom and the Broad Institute in the United States, using CRISPR gene editing technology to knock out about 18,000 genes in 1,000 human cancer cells, and find genes related to cancer growth or survival, so as to discover cancer treatment targets
.

The study found that CRISPR could not knock out 2 to 5 percent of these genes in these cancer cell lines, which is more common in cell lines derived from people of African descent and about
20 percent more than cell lines derived from other populations.
This means that in this "Cancer Dependency Atlas" initiative, some cancer treatment targets in people of African ancestry will be
missed.

In addition, there are studies that have shown that when treating sickle cell disease (a genetic disease that occurs mainly in people of African ancestry) with CRISPR gene editing, gRNAs designed according to the reference genome will not match the target genomic site, resulting in cutting genomic DNA at the wrong site, and this deviated editing of the target genomic locus may lead to potential risks
including cancer.

In the case of CRSIPR-Cas9, because the human genome varies by bloodline, the gRNA (red) sometimes does not match the target DNA sequence (yellow), resulting in the Cas9 enzyme (blue) not being able to complete the required cleavage and editing
.

Based on this research, the research team built a free online tool, Ancestrygarden.
org, to compare designed gRNAs with tens of thousands of genomes obtained from different populations to assess the impact
of ancestry problems on standard CRISPR-gRNA.

Ancestrygarden.
org

This research will prompt us to rethink how CRISPR technology is used in the lab, and the online tools developed will also help to better explore and understand variations in the human genome
.

Original source:

Sean A.
Misek， Aaron Fultineer， Jeremie Kalfon， et al.
Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries.
bioxiv，2022.