Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors

Abstract

Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

“…Type VI CRISPR-Cas systems include a single protein, Cas13 (formerly C2c2), that when assembled with a CRISPR-RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Shmakov et al, 2015; Smargon et al, 2017; Yan et al, 2018). Cas13 proteins are classified into distinct subfamilies (Cas13a-d), and all Cas13 proteins studied to date possess two enzymatically distinct RNase activities that are required for optimal interference (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). First, upon binding a precursor-crRNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al, 2016; East-Seletsky et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018).…”

“…Cas13 proteins are classified into distinct subfamilies (Cas13a-d), and all Cas13 proteins studied to date possess two enzymatically distinct RNase activities that are required for optimal interference (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). First, upon binding a precursor-crRNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al, 2016; East-Seletsky et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). Second, binding of an RNA target complementary to the crRNA (henceforth referred to as an activator-RNA) triggers Cas13 to cleave RNA non-specifically by activating the enzyme’s two HEPN-domains to form a single composite RNase active site (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Liu et al, 2017a; Liu et al, 2017b; Smargon et al, 2017; Yan et al, 2018).…”

“…First, upon binding a precursor-crRNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al, 2016; East-Seletsky et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). Second, binding of an RNA target complementary to the crRNA (henceforth referred to as an activator-RNA) triggers Cas13 to cleave RNA non-specifically by activating the enzyme’s two HEPN-domains to form a single composite RNase active site (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Liu et al, 2017a; Liu et al, 2017b; Smargon et al, 2017; Yan et al, 2018). This HEPN-activated conformation of Cas13 is a general nuclease, which is capable of cleaving either the RNA molecule that it was activated by ( cis cleavage), or any other RNA molecule that it happens to encounter ( trans / collateral cleavage).…”

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…Moreover, gene editing by Cpf1 results in lower off-target effects than Cas9, as evidenced by genome-wide analysis of edited cells [74]. Finally, the discovery of Cas13a and CasRx as RNA-guided nucleases targeting RNA paves the way to new therapeutic approaches based on RNA editing [69,75]. …”

CRISPR/Cas9: the gold standard of genome editing?

“…The tool, named CasRx, comes from the same family of CRISPR enzymes as Cas9 but has expanded the gene editing tool box from DNA to RNA [5]. In an experiment the team were able to correct a protein imbalance in a neuron exhibiting frontotemporal dementia (FTD).…”