The study introduces CRISPR-Cas13a as a powerful tool for RNA targeting and manipulation in mammalian and plant cells. Cas13a, a class 2 type VI RNA-guided RNA-targeting effector, was engineered for efficient RNA knockdown and binding. After screening fifteen Cas13a orthologs in E. coli, LwaCas13a from Leptotrichia wadei was identified as the most effective. LwaCas13a can be expressed in mammalian and plant cells for targeted RNA knockdown and binding. It provides comparable knockdown efficiency to RNAi but with significantly improved specificity. Catalytically inactive LwaCas13a retains RNA binding ability, enabling programmable tracking of transcripts in live cells. The study demonstrates that LwaCas13a can efficiently knockdown both reporter and endogenous transcripts in mammalian cells, with knockdown levels comparable to RNAi. It also shows that LwaCas13a can knockdown transcripts in plant cells, achieving over 50% knockdown for three genes. The study further shows that LwaCas13a can target nuclear transcripts, such as MALAT1 and XIST, with knockdown efficiencies up to 83.9% for XIST, which is more effective than position-matched shRNAs. The study also shows that LwaCas13a can be used for multiplexed gene knockdown, with a CRISPR array expressing multiple guides under a single promoter. It demonstrates that LwaCas13a can be used for in vivo imaging, with dCas13a (catalytically inactive Cas13a) enabling RNA binding and tracking. The study also shows that LwaCas13a has no sequence-specific off-target activity, as evidenced by transcriptome-wide RNA sequencing, which found no off-target effects in LwaCas13a conditions, despite comparable knockdown levels to shRNA. The study concludes that CRISPR-Cas13a is a versatile platform for RNA targeting with wide applicability for studying RNA in mammalian cells. It can be used for genome-wide pooled knockdown screening, interrogation of lncRNA and nascent transcript function, allele-specific knockdown, and RNA viral therapeutics. Additionally, dCas13a and its derivatives enable RNA pulldown to study RNA-protein interactions, imaging via reconstitution of split fluorophores, translational modulation, RNA base editing, epitranscriptomic perturbation, splicing modulation, or targeted induction of apoptosis based on RNA expression levels. The study suggests that Cas13a may be generally extended to other eukaryotic organisms, paving the way for a range of transcriptome analysis tools.The study introduces CRISPR-Cas13a as a powerful tool for RNA targeting and manipulation in mammalian and plant cells. Cas13a, a class 2 type VI RNA-guided RNA-targeting effector, was engineered for efficient RNA knockdown and binding. After screening fifteen Cas13a orthologs in E. coli, LwaCas13a from Leptotrichia wadei was identified as the most effective. LwaCas13a can be expressed in mammalian and plant cells for targeted RNA knockdown and binding. It provides comparable knockdown efficiency to RNAi but with significantly improved specificity. Catalytically inactive LwaCas13a retains RNA binding ability, enabling programmable tracking of transcripts in live cells. The study demonstrates that LwaCas13a can efficiently knockdown both reporter and endogenous transcripts in mammalian cells, with knockdown levels comparable to RNAi. It also shows that LwaCas13a can knockdown transcripts in plant cells, achieving over 50% knockdown for three genes. The study further shows that LwaCas13a can target nuclear transcripts, such as MALAT1 and XIST, with knockdown efficiencies up to 83.9% for XIST, which is more effective than position-matched shRNAs. The study also shows that LwaCas13a can be used for multiplexed gene knockdown, with a CRISPR array expressing multiple guides under a single promoter. It demonstrates that LwaCas13a can be used for in vivo imaging, with dCas13a (catalytically inactive Cas13a) enabling RNA binding and tracking. The study also shows that LwaCas13a has no sequence-specific off-target activity, as evidenced by transcriptome-wide RNA sequencing, which found no off-target effects in LwaCas13a conditions, despite comparable knockdown levels to shRNA. The study concludes that CRISPR-Cas13a is a versatile platform for RNA targeting with wide applicability for studying RNA in mammalian cells. It can be used for genome-wide pooled knockdown screening, interrogation of lncRNA and nascent transcript function, allele-specific knockdown, and RNA viral therapeutics. Additionally, dCas13a and its derivatives enable RNA pulldown to study RNA-protein interactions, imaging via reconstitution of split fluorophores, translational modulation, RNA base editing, epitranscriptomic perturbation, splicing modulation, or targeted induction of apoptosis based on RNA expression levels. The study suggests that Cas13a may be generally extended to other eukaryotic organisms, paving the way for a range of transcriptome analysis tools.