Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

Abstract

The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development

“…Three studies of Cas9-injected mouse zygotes explicitly suggest that MMEJ is responsible for repair, rather than C-NHEJ (Li et al, 2015; Yang et al, 2013; Yasue et al, 2014), because the same alleles were repeatedly recovered in independently derived mice and repaired alleles appeared to utilize microhomologous repeat sequences. We also found that the alleles in Cas9-injected rats (Shao et al, 2014) and monkeys (Niu et al, 2014) have the sequence features expected from microhomology-mediated repair.…”

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

“…Indeed, in the last year, several enhancers have been deleted in mice using 2c CRISPR including those associated with expression of Bcl11a , 94 Blimp1 , 95 Mef2c , 96 Stat5 , 97 Pcdh , 98 Mmp13, 99 Gata3 , 100 and Cebpa . 101 In each case, deletion of the enhancer compromised expression of the associated target gene.…”

Customizing the genome as therapy for the β-hemoglobinopathies

“…47,48,58 Introduction of 2 engineered nucleases can result in targeted deletion or inversion, duplication, local indels at nuclease cleavage sites, or translocations/ chromosomal rearrangements. [60][61][62][63][64][65][66][67][68][69][70][71][72][73] Here, we review genome-editing approaches for genetic correction and disruption strategies for the b-hemoglobinopathies as summarized in Figure 1 and Table 1.…”