Modified CRISPR Gene Editing Proves Highly Accurate

Scientists in South Korea have shown that a Cas9 gene editing variant has much fewer off-target effects.

AsianScientist (Apr. 24, 2017) – According to researchers at the Center for Genomic Engineering at the Institute of Basic Science (IBS), a modified form of CRISPR gene editing is accurate enough to identify and substitute just one nucleotide out of the three billion nucleotides of the human genome. These findings have been published in Nature Biotechnology.

“It is the first time that the accuracy of this base editor has been verified at the whole genome level,” explained study lead author Professor Kim Jin-Soo.

The recent rapid progress in gene editing tools has caused a frenzy excitement in the biology community. The main protagonist of the current third-generation DNA scissors is CRISPR, a tool that is quicker and cheaper than its predecessors. By cutting out a small DNA sequence, CRISPR-Cas9 and CRISPR-Cpf1 are used to silence or reduce the expression of faulty genes.

However, last year, a new base editor method that does not cause random DNA deletions and insertions, but instead replaces only one DNA base, attracted the biologists’ attention. These types of gene corrections are critical as several diseases are caused by the misspelling of one of the four basic components of DNA; adenine (A), cytosine (C), guanine (G), and thymine (T). Single-nucleotide errors in DNA are referred to as point mutations. Examples of diseases caused by point mutations include: cystic fibrosis, sickle cell anemia and color blindness.

Unlike the existing third-generation DNA scissors, the base editor method consists of a variation of CRISPR-Cas9 (nCas9, nickase) fused with another enzyme called cytosine deaminase, which replaces the DNA component C with T. The scissors are directed to the correct position on the DNA by a guide RNA. However, up to now, it was not known whether the base editor was working only in the area of the faulty gene or if it was unnecessarily substituting Cs in other areas (off-target).

Just one month after reporting the first successful base editing to modify a single nucleotide in dystrophin and tyrosinase genes in animals, the same team has demonstrated the accuracy of this method at the genome scale.

In order to verify the correctness ofgene editing over the entire genome, IBS researchers modified an error-checking technique known as Digenome-seq to adapt it to the base editor method. Digenome-seq was used and validated last year, when the team analyzed the accuracy of CRISPR-Cpf1 and Cas9. IBS researchers also improved the computer program (Digenome 2.0) to identify off-targets more comprehensively and compared different guide RNAs, to find the one that reduces malfunctions and increases specificity.

Using this technique, the team demonstrated correctness of the base editor technique and they found it to be even more accurate than the current third-generation CRISPR-Cas9. The base editing technique induced C-to-T conversions in 1-67 sites in the human genome, while CRISPR-Cas9 caused cleavages in 30-241 sites, meaning that the base editor is making less off-target changes.

“Therefore, it is expected that these base editors will be used as widely as the popular CRISPR technology,” said Kim.

The article can be found at: Kim et al. (2017) Genome-Wide Target Specificities of CRISPR RNA-guided Programmable Deaminases.


Source: Institute for Basic Science; Photo: Shutterstock.
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