Adaptive Immunity
The type of host defense system that CRISPR/Cas9 is in bacteria and archaea
What is an adaptive immune system?
The selectable marker on pCAS
What is KanMX?
The functional nickname assigned to the Cas9 enzyme
What is "molecular scissors"?
The two major pathways DNA uses to repair a double strand break from Cas9
What are non-homologous end joining (NHEJ) and homology-directed repair (HDR)?
The observable colony color of wild-type yeast
What is white?
The location in the host bacterial genome where ~20 bp fragments of foreign DNA are inserted to remember the infection
What are the palindromic repeats of the CRISPR locus?
The strong yeast promoter on pCAS that allows for high level of expression
What is RNR2 promoter?
What is gRNA?
The error-prone mechanism that causes random insertions or deletions, making gene editing hard to regulate
What is non-homologous end joining (NHEJ)?
The gene responsible for maintaining the normal color in wild-type (WT) yeast
What is ADE2?
The two specific RNA molecules that form the functional gRNA
What are crRNA and tracrRNA?
What is SV40 NLS?
The primary function of gRNA binding to Cas9 before cleavage
What is increasing the affinity between Cas9 and dsDNA so that Cas9 can indirectly bind dsDNA?
The repair mechanism that uses a repair template to allow for precise gene editing
What is homology-directed repair (HDR)?
The resulting genotype when ADE2 is successfully targeted and disrupted by CRISPR/Cas9
What is mutant ade2 yeast cells?
The ultimate action the Cas9-gRNA complex performs on the foreign DNA
What is a double strand break (DSB)?
The two origins of replication on pCAS, required for plasmid replication in bacteria and yeast, respectively
What are the pUC origin and 2 micron origin?
The specific action of Cas9 once anchored to the gRNA
What is creating a DSB in a specific target of DNA?
Similarity between NHEJ and HDR
What is that both pathways repair DNA after a DSB from Cas9?
The intermediate that accumulates in mutant ade2 yeast to produce a red pigment
What is AIR?
What is the CRISPR/Cas9 system acts as an adaptive immune system, where foreign DNA fragments are added to CRISPR repeats, crRNA and tracrRNA make gRNA which binds cas9 to make DSB?
The seven major components of pCAS
What are sgRNA, KanMX, RNR2 promoter, Cas9 ORF, SV40 NLS, pUC origin, and 2 micron origin?
The two elements of the Type II Cas9 system and the responsibility of each
What are gRNA (anchor that binds Cas9 and increases affinity between it and dsDNA) and Cas9 ("molecular scissors" that makes a DSB in target DNA)?
The shared action of NHEJ and HDR, as well as the key difference
What is repairing DNA after a Cas9 DSB, where NHEJ is error-prone and causes random indels and HDR uses a repair template for precise gene editing?
The reason ADE2 is used as a target sequence in this lab
What is ADE2 maintains white WT yeast, and targeting it leads to mutant ade2 genes which accumulate AIR to turn the yeast red?