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After Mycoplasma genitalium was sequenced in 1995, it was known to have the smallest genome and was thought to be a minimal cell at the time. However, the 1999 research suggested that Mycoplasma genitalium consists of many non-essential genes and can not be considered a minimal cell. This motivated JCVI scientists to develop a synthetic cell with minimal genome. In 2010, Mycoplasma mycoides JCVI syn1 was presented as the first self-replicating synthetic cell. Again, this synthetic cell also had few non-essential genes which required further gene reduction. At last, in 2016 a M. mycoides JCVI syn3 was introduced which only contains 473 genes.
A) Design-built-test (DBT) cycles to create JCVI-syn3.0, B) Comparison of JCVI-syn1.0 with JCVI-syn3.0
Problem Statement
149 genes of JCVI-syn3.0 have unknown functions.
Goal
Construct a genome-wide CRISPRi library in order to decipher the gene functionality.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system consists of a CRISPR-associated Cas9 enzyme which acts as a "molecular scissors" to cut the targeted DNA whereas a guide RNA is a type of RNA molecule which can bind to Cas9 and "guides" the Cas9 to a specific DNA location. The gene edits using CRISPR/Cas9 is precise and allows natural DNA repair to take over.
Unlike the CRISPR/Cas9 system, CRISPR mediated interference (CRISPRi) method is used for targeted gene regulation which includes catalytically inactive Cas9 protein lacking endonuclease activity. CRISPRi system can be used for targeted gene regulation.
CRISPR
CRISPRi
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