Lecture 35: CRISPR & Gene Editing Technologies Flashcards
What is genome-editing?
cut genome by “DNA scissors”, CRISPR/Cas9
can be used to remove change and add DNA
What are gene editing technologies?
a number of gene editing tools have been developed over the past 25 years
essentially all perform the same function but newer ones are easier to program
What is CRISPR/Cas9?
part of the prokaryotic immune system that defends against viruses
What are the components of CRISPR/Cas9 components?
Cas9 is recruited to the DNA target site by the duplex tracrRNA;crRNA (guide RNA)
the crRNA binds the complementary DNA strand upstream of the PAM sequence
What are the three key elements of CRISPR/Cas9?
the Cas9 DNA endonuclease
a crRNA, which contains a 20 bp sequence complementary to target
a trans-activating crRNA (tracrRNA), which acts as a bridge between the crRNA and the Cas9 enzyme
What is the mechanism of CRISPR/Cas9 DNA cleavage?
DNA endonuclease is targted to a DNA sequence via a single guide RNA (sgRNA) sequence, upstream of the protospacer-associated motif (PAM, resulting in a-bp double strand break (DSB) upstream of the NGG
the resulting DSBs are subsequently either by non-homologous end joining (NHEJ) or by homology-directed repair (HDR)
What is off-target DNA cleavage?
schematic of CRISPR/Cas9 off-target sites with 1-bp insertion (DNA bulge) or 1-bp deletion (RNA bulge)
the 20-nt guide sequence in the sgRNA is shown with genomic target sequence (protospacer) containing single-base DNA bulge or single-base sgRNAbulge
What are the steps of designing a crRNA spacer sequence?
- identify a PAM (NGG) sequence in the DNA sequence you would like to target
- determine the 5’ start of the actual sgRNA targeting sequence by counting 20 nucleotides upstream of the PAM sequence
- determine the actual sgRNA targeting sequence
How is CRISPR/Cas9 delivered?
Cas9 may be delivered as a DNA or mRNA molecule encoding for the cas9 gene, or it may be delivered as a functional ribonucleoprotein (RNP)
regardless of cargo format, the largest challenge lies in delivering the cargo across the cell emmebrane
a variety of viral and nonviral methods have been derived to achieve successful delivery across the cell membrane
What are the three elements required in base editing?
a Cas nickase or Cas fused to a deaminase that makes the edit
a gRNA targeting Cas to a specific locus
a target base for editing within the editing window specified by the Cas protein
What are prime-editors?
prime-editors use an engineered reverse transcriptase fused to Cas9 nickase and a prime-editing guide RNA (pegRNA)
What is CRISPR screening?
CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function
CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis
How is CRISPR used within in vivo therapy?
diseases of the eye and ear are already being treated in vivo (directly in the living eye) in clinical trials
How is CRISPR used within ex vivo therapy?
useful for blood diseases sickle cell anemia, hemophilia, CAR T cancer
What is somatic gene editing?
somatic cell: any cell in a living organism other than a reproductive cell not passed on to offspring
could involve many tissues: heart, muscle, lung, skin, brain, blood, etc.
