Background After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. Results We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. Conclusions Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

The anticancer and anti-inflammatory effects of carboxyamidotriazole (CAI) have been demonstrated in several studies, but the underlying mechanisms remain to be elucidated. This study showed that CAI caused metabolic reprogramming of pancreatic cancer cells. The inhibition of mitochondrial oxidative metabolism by CAI led to increased glutamine-dependent reductive carboxylation and enhanced glycolytic metabolism. The presence of environmental substances that affect cellular metabolism, such as glutamine and pyruvate, attenuated the anticancer efficacy of CAI. Based on the action of CAI: 1) when glutamine was removed, the NAD+/NADH ratio was decreased, the synthesis of cellular aspartate was reduced, and autophagy flux was blocked; and 2) when glycolysis was pharmacologically inhibited, the ATP level was significantly decreased, the cell viability was greatly inhibited, and the compensatory rescue effect of glutamine was eliminated. When combined with chemotherapy, cotreatment with CAI and the glycolysis inhibitor 2-deoxyglucose (2-DG) inhibited the pancreatic cancer progression after chemotherapy. As the inhibition of mitochondrial oxidative metabolism can explain several anticancer activities of CAI reported previously, including inhibition of calcium entry and induction of reactive oxygen species, we demonstrate that inhibition of mitochondrial oxidative phosphorylation may be the fundamental mechanism of CAI. The combination of CAI and 2-DG causes energy depletion in cancer cells, eliminating the rescue effect of the metabolic environment. Inhibiting pancreatic cancer progression after chemotherapy is a rational application of this metabolism-disturbing combination strategy.

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR/Cas9-mediated editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.