In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

Abstract

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

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].…”

“…HITI-mediated transgene knock-in occurs more preferentially in the forward than in the reverse direction because the forward-directed transgene knock-in alters the genomic gRNA target sequence to prevent additional CRISPR/Cas9 cutting. Suzuki et al demonstrated that HITI worked in HEK293 cells and postmitotic neurons, and that HITI introduced the wild-type exon to rescue visual function using a rat model of retinitis pigmentosa as a proof of concept of its potential use for gene correction therapy [68].…”

“…Notably, a novel NHEJ-mediated site-specific transgene insertion method named homology-independent targeted integration (HITI) has been reported [68]. In CRISPRObLiGaRe, a guide RNA (gRNA) target sequence located in the genome is added in the same orientation into the targeting vector [66,67].…”

“…In CRISPRObLiGaRe, a guide RNA (gRNA) target sequence located in the genome is added in the same orientation into the targeting vector [66,67]. In contrast, the targeting vector for HITI contains the gRNA target sequence in the opposite orientation to the genome [68]. HITI-mediated transgene knock-in occurs more preferentially in the forward than in the reverse direction because the forward-directed transgene knock-in alters the genomic gRNA target sequence to prevent additional CRISPR/Cas9 cutting.…”

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

“…In this regard, Suzuki et al have recently reported that CRISPR/Cas9 systems achieved knock-in into the target Tubb3 locus in non-dividing mouse primary neuronal cells by homology-independent targeted integration (HITI) using plasmid-mediated and AAV vector-mediated delivery. 36) However, the efficiency was not high possibly because of the nature of Cas9 that it generates DSB with blunt ends. DSB with blunt ends mainly allows homology-directed repairs (HDRs) which barely occur in non-dividing cells, 37) leading to the observed low efficiency.…”