Principles for rational Cas13d guide design

Abstract

<jats:title>Abstract</jats:title><jats:p>Type VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences.</jats:p><jats:p>We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 – 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the model’s generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.</jats:p>

Programmable RNA Targeting Using CasRx in Flies

“…Optimization of gRNA design may further improve these systems as CasRx gRNAs have been shown to have variable knockdown efficiency. 5,53 Nevertheless, this is an important first step towards making transcriptome engineering a viable in vivo technology and provides a foundation for future experiments to mitigate the off-target and toxic attributes of the enzyme to make a new, viable tool in the expanding gene-editing toolbox.…”

Programmable RNA Targeting using CasRx in Flies

“…For example, the phenotypes of y varied by their expression, with ubiquitous expression of CasRx resulting in a Ubiq-CasRx/+; gRNA y /+ lethal phenotype and embryo and wing and body specific expression mitigated lethality phenotype seen in Ubiq-CasRx expression. Optimization of gRNA design may further improve these systems as CasRx gRNAs have been shown to have variable knockdown efficiency [5,47]. Nevertheless, this is an important first step towards making transcriptome engineering a viable in vivo technology and provides a foundation for future experiments to mitigate the off-target and toxic attributes of the enzyme to make a new, viable tool in the expanding gene-editing toolbox.…”

Deep screening of proximal and distal splicing-regulatory elements in a native sequence context

“…The limitation of the one-vector system was also recently noted in an independent study 44 . To overcome this issue, we used two separate lentiviral vectors for dCas13d and gRNA expression, which were sequentially transduced into cells, similar to recent Cas13d-based knockdown screens published during the course of this study [42][43][44][45][46][47][48] . Another key issue that made dCas13d-based screening more challenging, compared to Cas13d-based knockdown screening, is likely the requirement for more stable binding of dCas13d/gRNA and target sites to effectively compete with cognate RNA-binding effectors.…”

“…Several studies indicated that gRNA length may affect the efficiency of Cas13d-mediated target RNA cleavage 42,45,47,56 . In our initial transfections, we used the 22-nt gRNA D [9,30] targeting the ISS-N1 region as a positive control segRNA.…”

“…Targeted cleavage of specific RNA transcripts by the Cas13 protein is achieved by using a guide RNA (gRNA) that consists of a direct repeat (DR) and a 22-30 nucleotide (nt) spacer that is complementary to the target RNA sequence. The Cas13 system has been used to perform high-throughput knockdown screenings of genes that influence cellular fitness, including by targeting circular RNAs and long noncoding RNAs [42][43][44][45][46][47][48] , through the use of a pooled lentiviral gRNA library. In addition, the catalytically dead Cas13 (dCas13) has been used as a programmable RNA-binding module, fused to various effector domains for applications in RNA editing 49 , m 6 A modifications 50 , modulation of splicing 37,51,52 and polyadenylation 53 , live cell RNA imaging 54 , and mapping of protein-RNA interactions through proximity ligation 55 .…”