Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas systems contain a single effector ribonuclease, Cas13, that binds and processes a CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can be engineered to target and modulate RNA processes in human cells, illustrating the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, editing, or imaging tool [6-8]. While Cas13 has been successfully used by several groups, our lab has observed significant variability in Cas13 KD ability depending which protocol is being followed [9-12]. To further understand this variability and generate a robust Cas13 KD protocol we thoroughly tested which Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA transfected, methods for analyzing KD efficiency, and report an optimized method for carrying out and analyzing Cas13 mediated RNA KD experiments. The method outlined in this paper illustrates a faster and more reliable protocol to iteratively test gRNA performance and target gene KD.
Keywords: CRISPR; Cas13; RNA knockdown; RNA-targeting; RfxCas13d; Type VI CRISPR-Cas systems.
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