Details of CRISPR knock out efficacy
CRISPR technology is used mainly to disrupt genes – to create so called knock outs. Insertions and deletions introduced into the sequence typically render the gene nonfunctional, as its protein is put out of the original reading frame (the order in which 3-base-long codons are read) and acquires premature termination codon (because the sequence is read in another frame). This intervention does not always lead to knock out, because of long-known mechanisms grouped under term: biological plasticity/rescue.
Cells try to alleviate results of mutations disrupting genes. There are known cases in the human population, where individuals carry knocking out mutations – with potentially fatal consequences – but the organism essentially rescues the original product by skipping mutated parts, editing transcripts, or reinitiating transcription. The main mechanism of disruption – nonsense mediated decay – has also its own rules and optimizations, such as omitting premature stop codons in the last exon of the gene. Pathways of biological rescue clearly influence the efficacy of CRISPR.
First study assessed effects of CRISPR knock outs in two cell lines (HAP1 and K562). After introducing deletions and frameshifts in 193 genes, the authors observed that 1/3 of genes is still expressed “at residual level”. They proposed a model, in which a protein is knocked out only after (in order): modified exon is not skipped, nonsense mediated decay is present, and translation is not reinitiated. The work is available here.
Second study verified details of nonsense-mediated decay in the context of CRISPR knock outs. On the basis of 10,000 tumor exomes and transcriptomes, they created a tool called NMDetective, which helps to assess efficacy of modifications. In their model, premature termination codon should be (in order): outside of the last exon, farther than 150 nucleotides from the start of the gene, reside in an exon shorter than 407 nucleotides, and be at least 50 nucleotides from exon junction. The publication is available here.
Both works prove that design of CRISPR editions requires specific rules and models.
First publication: Smits, A.H., Ziebell, F., Joberty, G. et al. Biological plasticity rescues target activity in CRISPR knock outs. Nat Methods 16, 1087–1093 (2019) doi:10.1038/s41592-019-0614-5
Second publication: Lindeboom, R.G.H., Vermeulen, M., Lehner, B., Supek, F. The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy. Nat Genet 51, 1645–1651 (2019) doi:10.1038/s41588-019-0517-5