CRISPR Technology - Powerful but with major limitations CRISPR-Cas9 DNA editing relies on the recognition (more specific

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Crispr Technology Powerful But With Major Limitations Crispr Cas9 Dna Editing Relies On The Recognition More Specific 1 (232.35 KiB) Viewed 76 times

CRISPR Technology - Powerful but with major limitations CRISPR-Cas9 DNA editing relies on the recognition (more specifically, hybridization) of a target DNA sequence by the guide RNA sequence that is complexed with the Cas9 enzyme. The length of the guide sequence is about 20 bases long. The physics/chemistry tells us that even partial hybridization (e.g. with as few as 12 bases) of the guide sequence would result in a stable DNA/RNA complex because the reaction is usually performed at or below 37 °C. This would result in many off-target cuttings in the genome! a) (5 pts) Given the size of the human genome (~3x10⁹ for a haploid), how many sites on the human genome (haploid) would a 12 consecutive bases on the guide RNA hybridize to by chance (assuming the sequence space is statistically random)? b) (5 pts) Once the cuts are made in the chromosomes, the broken DNA molecules can usually be repaired by the two endogenous/intrinsic DNA repair mechanisms in the cells. What are the two mechanisms and their end products (of the repaired DNA)? c) (10 pts) Use a simple diagram to illustrate how you would use CRISPR-Cas9 technology to replace a defective gene (on chromosome 11) that causes beta-thalassemia with a normal/functional copy of the gene. Elaborate on the guide sequences and their orientations, and potential methods for delivering the necessary molecular components required (normal copy of the gene and CRISPR-Cas9 DNA/protein) into the nuclei of the cells.