Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing

Abstract

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) genome-editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the gen-ome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long-standing goal of connecting genotypes, phenotypes and fitness. Cas9 system to date have not been performed in an explicit ecological context, we will review case studies, often from traditional model organisms, that demonstrate different applications of the technology that could be used in ecology and evolution. Drawing from these studies and our own experiences, we will highlight some of the considerations and challenges associated with the application of CRISPR/Cas9 to nonmodel organisms, along with potential strategies to circumvent these issues.

Finding the Genomic Basis of Local Adaptation: Pitfalls, Practical Solutions, and Future Directions

“…Historically, this has been performed with transgenic techniques or with RNAi (RNA interference; e.g., Van Tyne et al 2011; Prasad et al 2012 b ). With new genome modification methods (e.g., CRISPR/Cas9; reviewed in Bono et al 2015), allelic function can be tested in a wider range of organisms with a previously unachievable level of precision. Functional validation under field conditions is ideal but is likely impossible for most experimental systems due to logistical, legal, and ethical challenges.…”

CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator ofIGF2imprinting in tumors

“…dCas9 is a catalytically dead CRISPR Cas9 mutant, which is defective in DNA cleavage, but maintains the ability to bind to the gRNA-guided gene target [21, 27]. The binding specificity is determined by both gRNA-DNA base pairing and a short DNA motif (protospacer adjacent motif [PAM] sequence: NGG) juxtaposed to the DNA complementary region [28–30]. …”

Color phenotypes are under similar genetic control in two distantly related species ofTimemastick insect

“…), and genome editing methods in general (Bono et al. ), could help to facilitate the discovery of the specific gene or genes underlying these phenotypes.…”