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

Abstract

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

“…Parallel studies using two-component CRISPR genome editing (18) of the first exon of Lmod1 resulted in a 151-bp deletion ( Fig. 2 A and B).…”

“…Guide RNAs (SI Appendix, Table S4) targeted either to the paired CArG boxes or exon one of mouse Lmod1 were cloned into the BbsI site of pX330-U6 chimeric BB-CBh-hSpCas9 plasmid (42230; Addgene). The guide RNAs were in vitro transcribed using a HiScribe T7 mRNA synthesis kit (New England BioLabs) and purified before assessment on an Agilent Bioanalyzer as described (18). Mouse zygotes were microinjected into the cytoplasm with Cas9 mRNA (100 ng/μL) and each guide RNA (100 ng/μL each) as described previously (17,57).…”

What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine?

“…The objective of the session was to provide attendees with sufficient background regarding CRISPR-Cas9 genome editing so that they could then meaningfully explore and discuss how genome editing might be relevant to clinical practice. We (the authors) gave short presentations that covered: the basic concepts of CRISPR-Cas9 genome editing; how CRISPR-Cas9 has transformed the way in which mouse models of human physiology and disease are made, making the process far more rapid and efficient; 2 proof-of-concept studies demonstrating that CRISPR-Cas9 targeting of genes in the mouse liver can beneficially modify lipid traits, heralding possible one-shot, lifelong treatments for dyslipidemia and coronary heart disease; 3, 4 potential dangers of genome editing (e.g., unintended mutations) and strategies to reduce those dangers; 4, 5 potential clinical scenarios in which one might contemplate performing GGE, including the preemption of devastating genetic diseases in one’s offspring, the reduction of risk of common disorders such as coronary heart disease and Alzheimer disease, and the addition of desired non-medical traits (“enhancements”); the possibility of implementing GGE not in embryos but rather in germ cells such as sperm and oocytes, which might be less morally objectionable to some people; and potential adverse consequences of modifying the human germline, such as spreading susceptibility to certain diseases or exacerbating societal inequities. In particular, it was pointed out that although many commentators have noted that in vitro fertilization paired with pre-implantation genetic diagnosis can often avoid any need for GGE, there are situations where all unmodified embryos will yield offspring with disease—two parents with a recessive disorder such as sickle cell disease or cystic fibrosis, or a parent homozygous or compound heterozygous for dominant mutations such as the Huntington disease repeat expansion.…”

Challenges and Opportunities in Linking Long Noncoding RNAs to Cardiovascular, Lung, and Blood Diseases

“…32 This becomes particularly relevant when the question arises as to whether the lncRNA functions in cis to control gene expression. Here, two-component CRISPR (defined as Cas9 endonuclease and sgRNAs) 32 could be used to delete the promoter and first exon of the lncRNA.…”