In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

Abstract

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.

Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes

“… 10 , 31 In this study, two different guides were used simultaneously with the aim of deleting the intermediate sequence and restricting the range of genome modifications, as has been previously indicated. 12–14 However, it did not appear as a solution in zygotes, which is contrary to what has been described in adult cells. 12 , 32 , 33 The preferential pathway for DNA repair differs between embryonic and adult cells and, in addition, DNA repair is accelerated in embryonic cells compared with adult cells.…”

“…Furthermore, in an attempt to control the result of genome editing, pairs of gRNAs, instead of only one guide, have been used, which has resulted in a more precise genetic outcome in terms of controlled deletions. 12–14 This could be extremely useful to simplify progeny characterization when generating genetically modified mouse models.…”

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations

“…This strategy is obviously faced with certain limitations associated with the design of the CRISPR guides used to induce double chain breaks. These include [ 22 ] the breaking distance between both guides. The optimal distance is between 23–148 bp.…”

“…This resulted in the restoration of the frameshift of the F5 gene with an efficiency of 41% and in the generation of a functional FV cell line. The design of the guides and the PAM orientation influences the cleavage/splicing efficacy achieved by the NHEJ-PD strategy [ 22 ]. The guides used to induce the KO model (KO guide 5 and KO guide 1) ( Figure 1 B) were designed with the PAM sites facing the sequence to be deleted (in/in).…”

“…Several authors have studied the efficacy of precise deletion by NHEJ (NHEJ-PD) and analyzed how this process works when the guides are separated by 23–148 bp, in which case, a precise deletion is the most likely event. The use of two guides to delete a defined DNA fragment and alter the frameshift efficiency of the affected locus in a pseudo-controlled way could allow the generation of knockout models for the study of the biology of the cell, thus contributing to correcting pathologic mutations or generating cellular or animal models of rare diseases [ 21 , 22 ]. In this study, a FV-deficient cell model was generated via CRISPR/Cas9 gene editing with a similar mutation to the one exhibited by the studied patient (c.3279G>A, p.Trp1093*).…”