CRISPR Terms

Cas (CRISPR Associate protein)—Genes found to be associated with CRISPR sequences that encode the nuclease or helicase proteins needed to identify and cut the targeted DNA sequence.  The first Cas protein to have its activity characterized was Cas9 from Streptococcus pyogenes and it is currently the preferred Cas for CRISPR/Cas gene editing.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)—Repeating segments of DNA found in the prokaryote genome where each repeat is followed by unique “spacer” DNA sequences.  In 2005 it was discovered that the spacer sequences are identical to phage DNA.

crRNA (CRISPR targeting RNA)—The transcribed region of the unique “spacer” sequences found in CRISPR regions;  crRNA guides Cas proteins to foreign genetic  elements.  Requires tracrRNA for complexing with Cas proteins.

Dead Cas9 (dCas9) – Cas9 with point mutations in the RuvC and HNH nuclease domains that prevent nuclease activity.  dCas9 can still bind to the target site and has been used as an activator/repressor of genes.

Double Stranded Break (DSB)—A break in both strands of the DNA double helix that elicits one of two repair mechanisms, Homology Directed Repair (HDR) or Non Homologous End Joining (NHEJ).

gRNA (Guide RNA)—The result of fusing the crRNA and tracrRNA components of the CRISPR/Cas system into one RNA molecule to targeted a specific sequence of genomic DNA.  gRNAs are not found in nature.  The crRNA sequence is synthesized in order to target the desired genomic DNA while the tracrRNA sequence comes from bacterial sequences needed to complex with Cas proteins.  Guide RNA can also be referred to as a single guide RNA (sgRNA).

HDR (Homology Directed Repair)—DNA repair mechanism that uses DNA homology to repair a DSB.  This mechanism can be used to insert novel DNA sequences into a genome by flanking the desired sequence with regions homologous to the sequences flanking an induced DSB.

InDel (Insertion/Deletion)—The insertion or deletion of DNA bases during DNA repair or damage that can result in disruption of the open reading frame.

NHEJ (Non-Homologous End-Joining)—ADNA repair mechanisms that often results in small inserts or deletions of nucleotides at the DSB.  This technique has been used to knockout individual genes in organisms.

PAM (Protospacer Adjacent Motif)—Specific DNA sequence that must follow the target DNA sequence in order for Cas9 to bind and cut DNA.  Cas9 from Streptococcus pyogenes has a PAM sequence of NGG.  New and novel PAM sequences have been identified in other Cas-like proteins.

“Spacer” Sequence—Sequence found between palindromic repeats on the CRISPR locus encoding phage DNA or – in the case of engineered CRISPR gene targeting – sequences of the target of interest.

TracrRNA (Trans-Activating crRNA)—A key small RNA in the CRISPR mechanism.  This RNA maintains two functions, hybridizing with the crRNA repeat sequence and binding Cas protein. The TracrRNA contains a sequence complementary to the palindromic repeat section of the transcribed crRNAUpon duplexing of this palindromic region, the crRNA/tracrRNA complex can then bind to Cas proteins for DNA targeting and cleavage.

Target sequence—Genomic DNA targeted by the CRISPR/Cas system.  Typically the target sequence is 20 nucleotides long and is immediately followed by the PAM sequence.