Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Abstract

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

“…Subsequently, the roles of JHs diversified extensively in insect lineages, and JHs now play key roles in regulating development, metamorphosis, reproduction, diapause, behavior, and polyphenism in insects (59,60). Recent rapid advances in genome-editing tools facilitate the efficient editing of genes or genomes in both model and nonmodel organisms (61)(62)(63). Thus, we are hopeful that the present study will encourage further research into the molecular mechanisms of JH actions in diverse insect groups and other arthropods, thereby yielding a better understanding of how JHs have acquired antimetamorphic actions as well as other pleiotropic actions during the evolution of insects.…”

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

“…However, for most clinical treatments or precise modeling of diseases in vitro, it is essential to achieve high-frequency knock-in of the repair template with the desired variant. Notably, since microhomology-mediated end joining (MMEJ)-assisted gene knock-in named PITCh (Precise Integration into Target Chromosome) [103,104] and NHEJ-mediated sitespecific gene insertion, HITI [68], are both HDRindependent precise knock-in methods, these strategies may increase the utility of genome editing in human cultured cells. Other techniques to enhance gene knock-in include inhibition of NHEJ with small compounds or Cas9 protein accumulation in an S-and G 2 -phase-dependent manner [105][106][107][108][109].…”

Easi-CRISPR: Efficient germline modification with long ssDNA donors

“…Such methods are based, however, on perturbation of fundamental DNA repair processes and may be toxic. Non-toxic approaches include the use of circular dsDNA donors with built-in artificial guide sequences that are linearized inside the cell/embryo (11)(12)(13). The linearized donor DNA is then inserted at the genomic Cas9 cut site by cellular ligases.…”