NEW YORK – Using a cytosine base editor, researchers in China have developed a genome-wide gene function screening method, using CRISPR-based gene knockout that doesn't rely on double-stranded DNA breaks, or DSBs.
In a study published on Monday in Nature Biotechnology, the researchers noted that canonical CRISPR knockout screens rely on Cas9-induced DSBs to generate targeted gene knockouts, but that distortions produced by the DSBs are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted.
To solve this problem, they developed a DSB-independent, genome-wide CRISPR screening method, which they named iBARed cytosine base editing-mediated gene KO, or BARBEKO. The method uses cytosine base editors for genome-scale knockout screens by perturbing gene start codons or splice sites, or by introducing premature termination codons, and it's integrated with barcoded guide RNAs for improved screening quality and efficiency.
Using BARBEKO, the researchers constructed a cell library through lentiviral infection at a high multiplicity of infection, and achieved efficient and accurate screening results with a substantially reduced number of starting cells. Importantly, they found that in comparison with Cas9-mediated fitness screens, BARBEKO screens were not affected by DNA cleavage-induced cytotoxicity in HeLa-, K562-, or DSB-sensitive retinal pigmented epithelial 1 cells.
When they used BARBEKO in knockout experiments of gene function, the researchers found that the method yielded comprehensive lists of genes affecting cell proliferation, either positively or negatively. Importantly, they found that negative screening, which is usually more technically challenging, was much easier to accomplish with the new technique. In addition, gene-independent cytotoxicity triggered by Cas9-mediated cleavage often muddles the results of negative selection screens related to cell fitness, because the depletion level triggered by gene loss of function is generally modest — that was less of a problem with BARBEKO.
The researchers did note that newly optimized versions of cytosine base editors, or CBEs, were developed during the course of their research, with extended targeting scope via a flexible protospacer adjacent motif or an expanded activity window. This could be helpful to CBE-based library design with improved guide RNA quality and coverage, they added. About 1,700 genes are missing in the current version of the BARBEKO library because of the limited targeting scope of the CBE version they used. However, other CBE constructs with higher efficiency and fewer off-targeting in DNA and RNA could also be used.