CRISPR‐Cas Systems for the Study of Immune Function

Abstract

The ability to manipulate genomes has provided a wealth of knowledge about the structure and function of the immune system. For decades, genome manipulation has relied on transgenesis and homologous recombination to introduce or excise genetic material. The recent development of targetable DNA (deoxyribonucleic acid)‐modifying enzymes that allow the precise modification of any given locus within genomes of all species has transformed the way genome editing and engineering are performed. These targetable enzymes, in particular the clustered regularly interspaced palindromic repeats–CRISPR‐associated (CRISPR‐Cas) systems, have made it more accessible to generate genetically modified organisms in laboratories. In addition to genome editing and engineering, CRISPR‐Cas systems have been adapted to regulate gene expression, visualise genomic loci as well as RNA (ribonucleic acid) transcripts and modify DNA and DNA‐interacting protein markings.

Key Concepts

  • Forward and reverse genetics have played a key role in identifying genes involved in the development and function of the immune system.
  • CRISPR‐Cas/Cpf1 systems for genome editing use short RNA molecules to direct an endonuclease (Cas9 or Cpf1) to virtually any location within the genome and introduce DNA double‐strand breaks (DSBs).
  • The newly introduced DNA DSBs trigger DNA repair pathways and facilitate the introduction of random or engineered mutations.
  • Four pathways are involved in the repair of DNA DSBs; of them, the nonhomologous end joining (NHEJ) and homologous recombination (HR) are the most prominent.
  • Repair via the NHEJ pathway is error prone and can result in the random insertion or deletion of genetic material (indels).
  • Repair via the HR pathway is error free and uses the sister chromatid as a template. The HR pathway can be taken over by providing a repair template [homology‐directed repair (HDR) template] containing engineered mutations. These HDR templates can be used to introduce specific genetic modifications or repair disease‐causing mutations.
  • In addition, CRISPR‐Cas9 systems have been adapted to stimulate or repress gene expression, visualise chromosomal regions, modify DNA and DNA‐binding protein marking and track RNA molecules.
  • CRISPR‐Cas systems have been used to identify genes involved in the development and function of the immune system, ranging from innate to adaptive immunity as well as to model human diseases.

Keywords: base editing; clustered regularly interspaced palindromic repeats (CRISPR); CRISPR‐associated (Cas); CRISPR from Prevotella and Francisella 1 (Cpf1); genome editing; genome engineering; immunology; RNA‐guided endonuclease; targetable nuclease

Figure 1. Architecture of the class 2 type II and type V CRISPR‐Cas operons. (a) Type II CRISPR‐Cas operons contain four genes (arrows), a CRISPR array (dark‐grey rectangles and light‐grey triangles) and a tracrRNA (red arrow). The architecture of type II CRISPR‐Cas operons differs slightly by bacterial strain, but all operons encode the signature gene cas9 (also known as csn1), cas1, cas2 and cas4 (or csn2). The CRSIPR array does not represent the actual number of repeat and spacer sequences. (b) Type V CRISPR‐Cpf1 operons also contain four genes (arrows) and a CRSPR array (dark‐grey rectangles and light‐grey triangles). Depending on the bacterial strain, the architecture of type V CRISPR‐Cas operons differs slightly, but all operons encode the signature gene cpf1 as well as cas1, cas2 and cas4. The CRSIPR array does not represent the actual number of repeat and spacer sequences.
Figure 2. Schematic representation of SpCas9 and FnCpf1 and their adaptation for genome editing. (a) Schematics of SpCas9 and SpCas9 adapted for genome editing in mammalian cells. SpCas9 has been optimised for the human codon usage to facilitate expression in mammalian cells. A nuclear localisation signal has also been attached to the C‐terminus of the protein to facilitate nuclear translocation of SpCas9/sgRNA. The TracrRNA and crRNA have been fused together to form a single‐guide ribonucleic acid (sgRNA). Green, PAM sequence; red, RNA molecules; grey, SpCas9; black arrowheads, cleavage sites; Black ‘N’, protospacer; red ‘N’, Spacer. (b) Schematic representation of FnCpf1 and FnCpf1 adapted for genome editing in mammalian cells. FnCpf1 has been codon optimised to facilitate expression in mammalian cells and a nuclear localisation signal has been fused to the C‐terminal end of the proteins to facilitate translocation of FnCpf1/crRNA to the nucleus of mammalian cells. Green, PAM sequence; red, RNA molecules; Grey, FnCpf1; Black arrowheads, cleavage sites; Black ‘N’, protospacer; red ‘N’, Spacer.
Figure 3. CRISPR‐Cas systems for genome editing. (a) Genome editing with Cas9‐D10A nickases requires a pair of nickases to introduce a DSB (double‐strand break). (b) Genome editing using dCas9‐Fok1 requires a pair of dCas9‐Fok1 to introduce a DSB. A spacer of 13–17 bp between the 2 dCas9‐Fok1 is required to accommodate for the nonspecific endonuclease Fok1. (c) Genome editing using inducible Cas9 systems. A split SpCas9 fused to a photoinducible dimerisation system named magnet (positive magnet, pMag; negative magnet, nMag). Blue light induces dimerisation of pMag and nMag and promotes the formation of the split SpCas9‐sgRNa – DNA (deoxyribonucleic acid) complex. (d) Base editing can be achieved by fusing CRISPR‐dCas9 with the cytidine deaminase APOBEC‐XTEN and a uracil glycosylase inhibitor (UGI).
Figure 4. Targeting strategies commonly used to edit genomes with CRISPR‐Cas systems. (a) Gene inactivation by the insertion of DSBs within the exon of a gene. Resolution of the break by NHEJ can generate a nonsense mutation at the break site of a missense mutation and a frame shift that might also result in generation of a premature STOP codon downstream of the missense mutation and degradation of the transcript via the nonsense‐mediated mRNA decay pathway. (b) Insertion‐specific mutations by insertion of a DSB within a specific exon and coadministration of a donor template that is homologous to the targeted region but includes the desired mutations. Resolution of the break by HR will result in insertion of the point mutations. (c) Gene inactivation by inversion or deletion of an entire gene by inserting 2 DSBs. Resolution of the breaks by NHEJ can lead to inversion or complete deletion of the intervening segment. (d) Deletion of the chromosomal segment containing more than 1 gene by insertion of 2 DSBs. Resolution of the break can result in the inversion or complete deletion of the intervening segment. (e) Generation of conditional alleles by insertion of 2 DSBs within specific introns and coadministration of donor templates containing recombinase recognition sequences (i.e. loxP sites) flanked by sequences homologous to the target regions. Resolution of the DNA breaks will result in insertion of the recombinase recognition sites. (f) Insertion of large DNA elements by insertion of a single break and coadministration of a donor template homologous containing the DNA segment to be incorporated flanked by homology arms. (g) Engineering of chromosomal translocation by insertion of DSBs on two chromosomes. Resolution of the breaks can result in chromosomal translocation. Insertion of a loxP site on each chromosome, at the break points, can also be used to generate inducible translocations. Grey or blue boxes, exons; red boxes, stop codons; yellow box, missense mutation; green boxes, designed modification; orange boxes, indels; blue triangles, DNA recombinase recognition sequences (i.e. loxP sites); DSB, double‐strand break.
Figure 5. CRSIPR‐Cas applications beyond genome editing. (a) CRISPR‐spCas9 can be converted into site‐specific transcriptional activators by the fusion of dCas9 to transcriptional activators (TA) such as VP16/VP64 or p65 activation domains. Tiling of these site‐specific transcriptional devices can modulate gene expression. (b) CRISPR‐spCas9 can be converted into site‐specific transcriptional repressors by the fusion of dCas9 to transcriptional repressors (TR) such as KRAB or SID, which promote epigenetic silencing. Tiling of these site‐specific transcriptional repressors can modulate repression of gene expression. (c) CRISPR‐SpCas9 can also been converted into locus specific epigenetic marking enzymes by the fusion of dCas9 to DAN methylases such as DNMT3A. The fusion of dCAs9 to DNA demethylases or histone acetylases/deacetylases can promote site‐specific epigenetic modifications. (d) Live imaging of DNA loci using dCas9 fused to eGFP. Tiling of dCas9‐eGFP with several sgRNAs allows the visualisation of genomic loci. (e) Live imaging of the RNA transcript using dCas9 fused to an eGFP and a small oligonucleotide that provides a PAM sequence (PAMer).
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Pelletier, Stephane(Nov 2016) CRISPR‐Cas Systems for the Study of Immune Function. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0026896]