Novel miniature CRISPR–Cas13 systems from uncultivated microbes effective in degrading SARS-CoV-2 sequences and influenza viruses

Abstract

Recently emerging SARS-CoV-2 virus has caused a global pandemic, with millions of infections and over 200, 000 deaths1. However, development of effective anti-coronavirus treatments has lagged behind. Competitive co-evolution between microbes and viruses has led to the diversification of microbe’s CRISPR/Cas defense systems against infectious viruses2,3. Among class-2 single effector systems, Cas13 is effective in combating RNA phages4. Previous studies have discovered novel Cas9 and Cas12 systems from metagenomic sequence of natural microbes5-7. Here we report the identification of two additional compact Cas13 families from natural microbes that are effective in degrading RNA viruses in mammalian cells. Using metagenomic terabase data sets, we searched for previously uncharacterized Cas13 genes proximal to the CRISPR array with a customized computational pipeline, and identified two most compact families (775 to 803 amino acids) of CRISPR-Cas ribonucleases, named hereafter as CRISPR/Cas type VI-E and VI-F. Out of seven Cas13 proteins, we found that Cas13e.1 was the smallest and could be engineered for efficient RNA interference and base editing in cultured mammalian cell lines. Moreover, Cas13e.1 has a high activity for degrading SARS-CoV-2 sequences and the genome of live influenza A virus (IAV). Together with a minimal pool of 10 crRNAs, Cas13e.1 could target over 99% of all known 3,137 coronavirus genomes for achieving antiviral defense. Overall, our results demonstrated there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, thus potentially useful for preventing viral infection in humans such as COVID-19.

How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application

“…The Cas proteins have markedly different sizes (currently ranging from ~800 to 1700 aa) [ 71 , 72 ] and domain compositions (i.e., RucV versus HEPN-nuclease motifs). The smaller size of single-effector proteins facilitates their transfection into cells, both directly using protein transfection methods or through viral vectors (i.e., Cas13bt, d, e, and f [ 71 , 72 , 73 ]). Conversely, larger Cas proteins may have additional (beneficial) functions, such as the RNase modules that process pre-crRNA.…”

CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research

“…A subsequent study demonstrated that C2c2 is a programmable RNA-targeting Cas endonuclease, guided by a crRNA . After these pioneering studies, several groups have published detailed reports revealing the structure and functions of the C2c2, which was then named Cas13a. − Furthermore, certain other members of the Cas13 family such as Cas13b, Cas13c, Cas13d, Cas13e, and Cas13f have been identified and characterized. − …”

“…Programmable single-base RNA editors have been developed by fusing nuclease deficient Cas13 proteins with nucleoside deaminase domains (Figure B, Table ). Both adenine to inosine (A-to-I) and cytosine to uracil (C-to-U) single-base editing can be conducted using dCas13-based RNA base editors. ,,, These methods have been used to correct several disease-associated G-to-A and T-to-C mutations at RNA level. ,, …”

A short overview of CRISPR-Cas technology and its application in viral disease control

“…This system is a promising tool for the inhibition of broad coronavirus infections, relevant in light of the current COVID-19 pandemic (Abbott et al 2020). In a recent preprint, Xu et al describe Cas13e.1, part of a novel family of small type VI CRISPR-Cas systems, which similarly demonstrated high cleavage activity when targeted against SARS-CoV-2 and IAV genomes (Xu et al 2020). In another study, the PRRSV genome was completely knocked down in cell culture by the use of the CRISPR-Cas13b system (Cui et al 2020).…”