DGK and DZHK position paper on genome editing: basic science applications and future perspective

Abstract

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.

Keywords: Animal models; CRISPR/Cas; Genome editing.

Fig. 1

Principle CRISPR/Cas systems. NHEJ non-homologous end joining, HDR homology-directed repair, PAM protospacer-adjacent motif, Cas9 CRISPR-associated protein carrying two nuclease domains, associated genes, dCas9 catalytically dead Cas9, Cas9 nickase a Cas9 carrying only one nuclease domain to induce single-strand breaks, sgRNA single-guide RNA, pegRNA prime editing guide RNA, VP64 gene inducer protein domain, KRAB gene suppressor protein domain

Fig. 2

CRISPR/Cas screening possibilities. Three strategies have been developed for CRISPR/Cas screens: in array screens, single wells/dishes of cells are infected with one individual sgRNA each, the readout is typically on cellular signature signals, such as comparing bulk transcriptomes or performing surface protein expression profiles. Pooled screens involve transducing several to many sgRNAs and applying a positive or negative selection on transduced cells. Target genes that generate the desired phenotype are uncovered by deep sequencing and subsequent ranking of measured sgRNAs. Third, cells expressing any kind of CRISPR/Cas machinery are transduced with a pooled sgRNA library. Cells will express a functional sgRNA copy together with a second copy of the sgRNA that allows identification by sequencing [barcoding, poly(A)-tailing, e.g., used in, e.g., Perturb-Seq, CROP-Seq etc.]. After droplet sequencing, sgRNA-mediated perturbation can be analyzed in single cells. Multiple sgRNAs against a target gene are used to validate the phenotype

Fig. 3

Domains of gene editing in animals. Whereas Cas9-mediated germline gene editing now becomes the standard technology for the generation of transgenic fish and rodents, larger mammals, like transgenic pigs are still generated by “conventional” technology. Pigs and dogs are important laboratory animals in the translational avenue to established somatic gene editing for clinical use

Fig. 4

Split-Intein-AAV-System: due to the packaging limit of adeno-associated virus (AAV), the most-frequently used Cas9 genes together with two guide RNA cannot be transducted by a single AAV. In the Split-Intein-System, the cell is infected with two different viruses, both containing one part of Cas9 and one part of the Intein gene. Expression of both constructs yields a complete, enzymatically active Cas9

Fig. 5

Concepts of human genome editing