On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

Abstract

Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

“…Our new data are in line with recent publications where genome engineering resulted in unexpected larger rearrangements [ 15 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. Our findings support the observations of Boroviak and colleagues [ 16 ]; however, the high-frequency occurrence of inverted re-insertions using the dual sgRNA approach has not previously been described in this context.…”

“…Our own research has observed frequent larger-than-expected deletions, potentially generated by the microhomology mediated end-joining (MMEJ) repair pathway [ 18 ]. Additionally, more recent findings report serial head-to-tail insertions of donor DNA templates [ 19 ]; error-prone repair pathways inserting unwanted deletions and insertions [ 20 ]; unintended ON-target chromosomal instabilities [ 21 ]; harmful chromosomal deletions [ 22 ]; and deleterious ON-target effects when aiming at homology directed repair (HDR) [ 23 ]. Hence, it is crucially important for precise model validation to know which outcomes CRISPR/Cas9 genome engineering can generate besides the actual aimed-for edit [ 24 ].…”

Erythrocytes 3D genome organization in vertebrates

“…The mice were maintained on a 12‐hour light/dark cycle with ad libitum food and water in a conventional animal facility 27 . All experiments were conducted at the Department of Experimental Animal Genetic Resources at the Institute of Cytology and Genetics, SB RAS (RFMEFI61914X0005 and FMEFI61914X0010).…”

Pannexin 1 Transgenic Mice: Human Diseases and Sleep-Wake Function Revision

“…Then, after purification, the DNA template was used for in vitro gRNA transcription using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S, Ipswitch, MA, USA). The gRNA was purified on RNA Clean and Concentrator-25 columns (Zymo Research, R1017, Irvine, CA, USA) [ 53 ]. SpCas9 messenger RNA was purchased from a commercial manufacturer (GeneArt CRISPR Nuclease mRNA, Thermo Fisher Scientific, Shanghai, China).…”