Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

Abstract

CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

Machine learning based CRISPR gRNA design for therapeutic exon skipping

“…SkipGuide is evaluated on gRNAs that lead to DNA double-stranded breaks in a small window (6-bp) surrounding and including the AG splice site acceptor motif. End-joining repair at cut sites outside of this region [34][35][36][37], and ablations of an exon's splice donor sequence [37,38], have been shown to also lead to exon skipping. Hence, modelling these cases can be future extensions to SkipGuide.…”

Effective downregulation of BCR-ABL tumorigenicity by RNA targeted CRISPR-Cas13a

“…However, García-Tuñón et al , in their other research article on CRISPR, published recently, highlighted the limitation of CRISPR/Cas9 in effectively knocking out target DNA without a null variation inducing homology directed repair template in the construct. They went on to demonstrate that effective knockdown could be achieved if sgRNAs target the splice donor site of the gene 31 . Moreover, it has been shown that in CML, using a DNA targeting CRISPR would mean designing custom guide RNAs per patient as the breakpoints might differ per case at the DNA level 10 .…”

Future Approaches for Treating Chronic Myeloid Leukemia: CRISPR Therapy

“…In the last five years, the number of scientific papers reporting work on CRISPR/Cas9 in the context of leukemia research has increased enormously [ 84 , 93 , 94 , 95 , 96 ]. Many of them concern in vitro studies to clarify the role of a variety of genes in leukemia development [ 97 ].…”