Introduction
Since it was first used to edit the mammalian genome,1 the targetable gene editing tool CRISPR (clustered regularly interspaced short palindromic repeats) has become widely accessible to researchers. Compared to older gene editing technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR has significant advantages: it is more efficient, faster to set up, and can be multiplexed: several DNA loci can be targeted in one experiment.2 CRISPR’s potential in neuroscience ranges from investigating fundamental processes underlying brain function and development, to modelling neurological diseases in both animals and cells, and perhaps to CRISPR-based therapies. This review discusses CRISPR’s current applications in cell and animal models aiming to clarify brain function and dysfunction, and some of the challenges that currently limit CRISPR’s use in neuroscience.
What is CRISPR?
The CRISPR gene editing system has been identified in and is derived from part of the prokaryote adaptive immune system, which defends against invading viruses or plasmids by specifically cleaving exogenous DNA. Adapting CRISPR for gene editing exploits the ability of CRISPR nucleases to make predictable DNA breaks at specifically targeted sequences. There are three types of CRISPR system (I, II and III) of which type II is most widely used in gene editing (see figure 1 below).
Figure 1. Using the Cas9 CRISPR system for gene editing. Guide RNA (gRNA) is composed of a scaffold sequence (required for binding between Cas9 and the gRNA) and a 20 base pair sequence which is designed complementary to the DNA target. The DNA target must be upstream of a proto-spacer adjacent motif (PAM) sequence, which is required for DNA cleavage. Cas9 nuclease is directed to the target DNA by base pairing between the target DNA and gRNA, resulting in a double-stranded DNA break. The double-stranded break can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). NHEJ results in either restoration of the wild-type sequence, insertions or deletions (indels). Indels vary in length and can cause frame-shift mutations, leading to premature stop codons and gene knockout. HR requires a DNA template with homologous regions up- and down-stream of the break. Therefore, a DNA repair template can be designed in order to introduce precise insertions, deletions or point mutations.
Figure 1. Using the Cas9 CRISPR system for gene editing.
Guide RNA (gRNA) is composed of a scaffold sequence (required for binding between Cas9 and the gRNA) and a 20 base pair sequence which is designed complementary to the DNA target. The DNA target must be upstream of a proto-spacer adjacent motif (PAM) sequence, which is required for DNA cleavage. Cas9 nuclease is directed to the target DNA by base pairing between the target DNA and gRNA, resulting in a double-stranded DNA break. The double-stranded break can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). NHEJ results in either restoration of the wild-type sequence, insertions or deletions (indels). Indels vary in length and can cause frame-shift mutations, leading to premature stop codons and gene knockout. HR requires a DNA template with homologous regions up- and down-stream of the break. Therefore, a DNA repair template can be designed in order to introduce precise insertions, deletions or point mutations.
First, guide RNA is designed complementary to a DNA target. Next, the guide RNA complexes with Cas9 nuclease (the CRISPR effector), followed by base-pairing between the guide RNA and its DNA target, which directs Cas9 to cleave the DNA. Finally, double stranded DNA breaks are repaired by either non-homologous end-joining (NHEJ) or homologous recombination (HR). NHEJ makes insertion or deletion mutations (indels) of varying lengths, usually resulting in a premature stop codon and gene knockout. Alternatively, DNA with the desired insertion, deletion or point mutation can be introduced to act as a repair template during HR, leading to precise mutations in the DNA.3
Cas9 is a nuclease, but CRISPR is not limited to nuclease activity. Inactivation of both catalytic domains in dead (d)Cas9 renders the nuclease unable to cleave DNA, but it can prevent transcription by steric hindrance in CRISPR interference (CRISPRi). CRISPRi can be enhanced by complexing dCas9 to repressors, for reversible gene knockdown, whereas dCas9-activator complexes can be used for reversible overexpression. In addition, dCas9 complexed to epigenetic modifiers can be used for methylation or histone modifications, and Cas9 with a fluorescent molecule can tag genomic loci.4 The range of functional domains coupled to Cas9 is expanding, linking CRISPR to advances in our understanding of genetic processes and our ability to manipulate them. One area where CRISPR has been readily adopted is in modelling neurological disease with human induced pluripotent stem cells (hiPSCs).
More at
http://www.acnr.co.uk/2017/02/crispr-for-gene-editing-in-neuroscience-and-neurological-disease/