L07 Chem 481 Lecture Notes - Lecture 23: Crispr, Cas9, Transcription Activator-Like Effector Nuclease
13 Dec 2016
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24 October 2016
Lecture 23: CRISPR-Cas Genomic Engineering
I. Genomic Engineering
A. Overview
1. Genomic Engineering
a. Refers to the process of making targeted modifications of the genome, epigenetic
markers, or outputs of genomic processing
b. This holds many implications in biotechnology, medicine such as for site-specific drug
development and other medical therapeutics
2. Strategies
a. Double strand break followed by:
• NHEG
• Or Homology-directed repair
3. Pre-CRISPR-cas
a. Goal = to create double strand breaks (DSB)
b. Zinc-finger nucleases (ZFN’s) and transcription activator-like effector nucleases
(TALENs) were used
c. These methods were expensive, time consuming (to develop/prepare) and had to be
sequence specific
B. CRISPR-cas
1. Clustered regularly interspaced short palindromic repeats (CRISPR)
a. Researchers have been studying since 1980s, but huge increase in use and funding since
2012
2. In Native Setting
a. CRISPR-cas is part of the bacterial “immune response” to bacteriophages
b. Incorporates foreign DNA from bacteriophages into the genome to defend against future
attacks by similar viruses
c. Many different types and classes of CRISPR-cas, same general principal
• Type II is main focus and most studied
d. 3 Main steps: spacer acquisition, CRISPR RNA biogenesis, interference
3. Interference
a. Protospacers and short palindromic repeats are identified in dsDNA and transcribed into
pre-crRNA (CRISPR RNA)
b. RNaseIII, trans-activating crRNA (tracrRNA) and Cas9 create a pre-cRNA-tracrRNA-
Cas9-RNaseIII complex
c. Then cuts at each spacer to create multiple crRNA-tracrRNA-Cas9 complexes
d. Complex recognized the dsDNA target at the proto-spacer target sequences and the proto-
spacer adjacent motif (PAM very important for recognition)
e. Then the complex cuts the dsDNA target to create a DSB
4. The Proto-spacer Adjacent Motif (PAM)
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