CRISPR History and Background
Figure 1: The current genome engineering technologies allow scientists to introduce double stranded breaks at specific sequences. Learn more on Addgene's Genome Engineering page .
2013 was an exciting time for genome engineering technology. A new system offered the first alternative to the current protein-based targeting (TALEN and Zinc Finger) methods used to specifically target a gene (or other DNA sequence). This new system uses a short RNA to guide a nuclease to the DNA target. This is CRISPR technology ( Figure 1 ).
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. So far three distinct bacterial CRISPR systems have been identified, termed type I, II and III. The Type II system is the basis for the current genome engineering technology available and is often simply referred to as CRISPR.
The crRNA targeting sequences are transcribed from DNA sequences known as protospacers. Protospacers are clustered in the bacterial genome in a group called a CRISPR array. The protospacers are short sequences (~20bp) of known foreign DNA separated by a short palindromic repeat and kept like a record against future encounters. To create the CRISPR targeting RNA (crRNA), the array is transcribed and the RNA is processed to separate the individual recognition sequences between the repeats. In the Type II system, the processing of the CRISPR array transcript (pre-crRNA) into individual crRNAs is dependent on the presence of a trans-activating crRNA (tracrRNA) that has sequence complementary to the palindromic repeat. When the tracrRNA hybridizes to the short palindromic repeat, it triggers processing by the bacterial double-stranded RNA-specific ribonuclease, RNase III. Any crRNA and the tracrRNA can then both bind to the Cas9 nuclease, which then becomes activated and specific to the DNA sequence complimentary to the crRNA ( Figure 2 ).
There are some restrictions to this new genome engineering system. Any potential genomic target sequence must have a specific sequence on its 3’ end (the protospacer adjacent motif, PAM). The PAM is present in the DNA to be degraded but not on the crRNA that will target it. The Type II CRISPR system is currently limited to target sequences that are ~20 nucleotides followed by NGG, the PAM sequence. Additionally, it is hypothesized that certain target sequences are believed to be problematic due to the RNA secondary structure they form. Interactions between the Cas9, tracrRNA and crRNA secondary structures are still poorly understood.
Figure 2: An overview of the endogenous Type II bacterial CRISPR/Cas system. Within the bacterial genome, a CRISPR array contains many unique protospacer sequences that have homology to various foreign DNA (e.g. viral genome). Protospacers are separated by a short palindromic repeat sequence. (A) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (B) The pre-crRNA is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit the RNAse III and Cas9 enzymes, which together separate the individual crRNAs. (C) The tracrRNA and Cas9 nuclease form a complex with each individual, unique crRNA. (D) Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complimentary to the crRNA. In the Type II CRISPR system a potential target sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly after where the crRNA would bind. (E) After the complex binds, the Cas9 separates the double stranded DNA target and cleaves both strands after the PAM. (F) The crRNA:tracrRNA:Cas9 complex unbinds after the double strand break.
RNA-guided human genome engineering via Cas9. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. Science . 2013 Feb 15;339(6121):823-6.PubMed.
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. Science . 2012 Aug 17;337(6096):816-21.PubMed.
Efficient genome editing in zebrafish using a CRISPR-Cas system. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Nat Biotechnol . 2013 Mar;31(3):227-9.PubMed.
RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. Nat. Biotechnol . 2013 Mar;31(3):233-9.PubMed.
Multiplex genome engineering using CRISPR/Cas systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science . 2013 Feb 15;339(6121):819-23.PubMed.