Ayush Noori | EduSTEM Breakthrough Biology
Introduction to CRISPR/Cas9:
CRISPR systems are bacterial defense mechanisms against viral invasion. Researchers have adapted CRISPR to perform potent and specific genetic editing across the diverse genomes of life. The CRISPR system consists of the CRISPR array, associated cas (CRISPR-associated) genes (including Cas9, Cas1, Cas2, and Csn2 genes), and the transactivating CRISPR RNA (tracrRNA) gene which has homology to the CRSIPR short palindromic repeats . The Cas9 endonuclease has two distinct lobes: the alpha-helical recognition (REC) lobe and the nuclease (NUC) lobe (which contains the HNH, RuvC, and C-terminal domains) [2, 3].
Protospacer Integration and Cas9 Complex Formation:
Figure 1. “CRISPR–Cas9-mediated DNA interference in bacterial adaptive immunity.” Please see Jiang and Doudna, 2017 .
In bacteria, CRISPR integrase – a heterohexameric complex of four Cas1 and two Cas2 proteins – along with the accessory histone-like protein Integration Host Factor (IHF), inserts protospacer sequences from invading viral DNA between short palindromic repeat sequences, forming the CRISPR array . Upon repeat infection, the CRISPR array is transcribed to yield precursor CRISPR RNAs (pre-crRNAs). Enzymatic cleavage of the pre-crRNAs by RNase III is facilitated by the tracrRNA and produces mature CRISPR RNAs (crRNAs), which consist of a variable protospacer and part of the CRISPR repeat sequence . The crRNA hybridizes with the tracrRNA to form the guide RNA complex . The ribose-phosphate backbone of the guide RNA complex subsequently complexes with the three alpha-helical domains of the REC lobe of Cas9 nuclease, causing a significant conformational shift in Cas9 to form the active crRNA-tracrRNA-Cas9 complex .
PAM Sequence Interrogation:
Figure 2. “The GG dinucleotide of the PAM is read out by major-groove interactions.” Base-specific hydrogen bonding between the PAM sequence (dt1, dG2, and dG3) and two arginine residues (Arg1333 and Arg1335) in the β-hairpin of the Cas9 C-terminal domain (red). Please see Anders et al. , 2014 .
The active crRNA-tracrRNA-Cas9 complex subsequently interrogates the genome for a 20-nucleotide target sequence complementary to the protospacer sequence of the crRNA, however this target sequence must lie immediately upstream (towards the 5’ end) of a specific protospacer adjacent motif (PAM) sequence (two to five nucleotides in length) . By only cleaving sequences upstream of the PAM sequence, the CRISPR system ensures that the native CRISPR array (which does not contain PAM sequences) is not cleaved by Cas9. In the most prevalent type II CRISPR system from Streptococcus pyogenes , the PAM sequence is 5’-NGG-3’ (where N can be any of the four DNA bases), however various other Cas9 proteins have other PAM sequences [7, 8]. Cas9 first searches for PAM sequence homology. Base-specific hydrogen bonding between the PAM sequence and two arginine residues in the β-hairpin of the Cas9 C-terminal domain destabilizes the target DNA duplex, prompting formation of an R-loop and enabling Watson-Crick base pairing between the crRNA and target sequence .
Target DNA Cleavage:
Complete hybridization between the crRNA and the target sequence causes (the HNH domain of) Cas9 to adopt a stable conformation and introduce a double stranded break (DSB) in the target sequence three nucleotides upstream of the PAM . The DSB is caused by the two nuclease domains of Cas9: an HNH-like domain that cleaves the DNA strand complementary to the crRNA sequence (target strand), and a RuvC-like domain that cleaves the strand opposite to the complementary strand (nontarget strand) [2, 3]. The DSB can then be repaired by nonhomologous end joining (NHEJ), which is prone to loss-of-function mutations and results in gene knockout, or homology directed repair (HDR), where a homologous donor DNA repair template is provided along with the CRISPR system and is used to repair the DSB [10, 11]. Via the introduction of a therapeutic donor DNA template, HDR allows the correction of deleterious mutations in the target sequence.
Figure 3. “Genome editing using double-stranded breaks (DSBs).” The DSB can be repaired by NHEJ, which is prone to loss-of-function mutations and results in gene knockout, or homology directed repair (HDR), where a therapeutic donor template allows for replacement of deleterious DNA. Please see Komor et al. , 2017 .
Figure 4. “Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features.” This diagram depicts the engineered sgRNA with the desired 20 nucleotide protospacer sequence versus the original bacterial function. The two “scissors” are the HNH and RuvC domains of Cas9. Please see Jinek et al. , 2017 .
This system is nearly identical in engineered scientific applications, however the crRNA and tracrRNA are fused to yield a single-guide RNA (sgRNA) with the desired protospacer sequence which complexes with Cas9 and performs the same function as the crRNA-tracrRNA complex.
- Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017 May 22;46:505-529. doi: 10.1146/annurev-biophys-062215-010822. Epub 2017 Mar 30.
- Jinek M, Jiang F, Taylor DW, et al. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 2014 Mar 14; 343(6176): 1247997. Published online 2014 Feb 6. doi: 10.1126/science.1247997
- Nishimasu H, Ran FA, Hsu PD, et al. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell. 2014 Feb 27; 156(5): 935–949. Published online 2014 Feb 13. doi: 10.1016/j.cell.2014.02.001
- Wright AV, Liu J-J, Knott GJ, Doxzen KW, Nogales E, Doudna JA. Structures of the CRISPR genome integration complex. Science. 2017 Sep 15; 357(6356): 1113–1118. Published online 2017 Jul 20. doi: 10.1126/science.aao0679
- Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III. Nature. 2011 Mar 31; 471(7340): 602–607. doi: 10.1038/nature09886
- Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17; 337(6096): 816–821. Published online 2012 Jun 28. doi: 10.1126/science.1225829.
- Leenay RT and Beisel CL. Deciphering, communicating, and engineering the CRISPR PAM. J Mol Biol. 2017 Jan 20; 429(2): 177–191. Published online 2016 Dec 1. doi: 10.1016/j.jmb.2016.11.024
- Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014 Sep 25; 513(7519): 569–573. Published online 2014 Jul 27. doi: 10.1038/nature13579
- Jiang F, Taylor DW, Chen JS, et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science. 2016 Feb 19; 351(6275): 867–871. Published online 2016 Jan 14. doi: 10.1126/science.aad8282
- Sander JD and Joung JK. CRISPR-Cas systems for genome editing, regulation and targeting. Nat Biotechnol. 2014 Apr; 32(4): 347–355. Published online 2014 Mar 2. doi: 10.1038/nbt.2842
- Komor AC, Bardran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017 Jan 12; 168(1-2): 20–36. Published online 2016 Nov 17. doi: 10.1016/j.cell.2016.10.044
With these courses, we hope to further our mission to make high-quality STEMX education accessible for all. For questions or support, please feel free to reach out to me at firstname.lastname@example.org.
EduSTEM Boston Chapter Founder
The premier source of past and present medical literature. Most supplemental information in Extensions is available via PubMed. When searching PubMed, be sure to use the “Free full text,” and “Sort by: Best Match” filters to find relevant and accessible results.
A large database of useful 3D structures of large biological molecules, including proteins and nucleic acids. Use the search bar to find a molecule of interest, which can then be examined using the Web-based 3D viewer.