How does CRISPR/Cas work?
The CRISPR/Cas system uses a three-phase process to confer immunity to invading genetic elements to the host cell: adaptation, expression, and interference. In the adaptation phase, short DNA fragments homologous to foreign DNA sequences are incorporated into CRISPR loci as “spacers”. Each of these integration events is accompanied by the duplication of a repeat sequence, creating a new spacer-repeat unit.1,2
In the expression phase, the CRISPR sequence containing the newly integrated spacers is transcribed, creating a long precursor CRISPR RNA (pre-crRNA) sequence. This long sequence is subsequently processed into short crRNAs via different mechanisms depending on the CRISPR/Cas system type present. Type I systems feature Cas6 cleavage of CRISPR-associated complex for antiviral defense (Cascade) complex-bound pre-crRNA, resulting in crRNAs containing an 8-nucleotide repeat fragment on the 5′ end and a hairpin structure containing the remainder of the repeat fragment on the 3′ flank. In Type II systems, a small trans-encoded RNA (tracrRNA) pairs with the repeat fragment of the pre-crRNA, guiding RNase III cleavage (in the presence of Cas9) within the repeat fragment sequence. In type III systems, Cas6 cleavage generates crRNAs from the pre-crRNA sequence, but these crRNAs are passed to Csm (Type III-A) or Cmr (Type III-B) complexes for further processing at the 3′ end.1,2
Finally, during the interference phase, the crRNAs guide Cas protein complexes to viral or plasmid target sequences matching that present in the initial spacers, resulting in degradation of the foreign genetic material. The exact mechanism varies depending on the host organism, but CRISPR/Cas systems are capable of targeting either foreign DNA or mRNA.2 For more information on CRISPR please navigate to our resource center here.
References:
1. D. Rath, et al., “The CRISPR-Cas immune system: biology, mechanisms and applications,” Biochimie 117:119-128, 2015.
2. K.S Makarova, et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol 9(6): 467-477, 2011.
In the expression phase, the CRISPR sequence containing the newly integrated spacers is transcribed, creating a long precursor CRISPR RNA (pre-crRNA) sequence. This long sequence is subsequently processed into short crRNAs via different mechanisms depending on the CRISPR/Cas system type present. Type I systems feature Cas6 cleavage of CRISPR-associated complex for antiviral defense (Cascade) complex-bound pre-crRNA, resulting in crRNAs containing an 8-nucleotide repeat fragment on the 5′ end and a hairpin structure containing the remainder of the repeat fragment on the 3′ flank. In Type II systems, a small trans-encoded RNA (tracrRNA) pairs with the repeat fragment of the pre-crRNA, guiding RNase III cleavage (in the presence of Cas9) within the repeat fragment sequence. In type III systems, Cas6 cleavage generates crRNAs from the pre-crRNA sequence, but these crRNAs are passed to Csm (Type III-A) or Cmr (Type III-B) complexes for further processing at the 3′ end.1,2
Finally, during the interference phase, the crRNAs guide Cas protein complexes to viral or plasmid target sequences matching that present in the initial spacers, resulting in degradation of the foreign genetic material. The exact mechanism varies depending on the host organism, but CRISPR/Cas systems are capable of targeting either foreign DNA or mRNA.2 For more information on CRISPR please navigate to our resource center here.
References:
1. D. Rath, et al., “The CRISPR-Cas immune system: biology, mechanisms and applications,” Biochimie 117:119-128, 2015.
2. K.S Makarova, et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol 9(6): 467-477, 2011.
