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Intrinsic targeting of host RNA by Cas13 constrains its utility

Nature Biomedical Engineering volume 8, pages 177–192 (2024)Cite this article

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Abstract

Cas13 can be used for the knockdown, editing, imaging or detection of RNA and for RNA-based gene therapy. Here by using RNA immunoprecipitation sequencing, transcriptome profiling, biochemical analysis, high-throughput screening and machine learning, we show that Cas13 can intrinsically target host RNA in mammalian cells through previously unappreciated mechanisms. Different from its known cis/trans RNA-cleavage activity, Cas13 can also cleave host RNA via mechanisms that are transcript-specific, independent of the sequence of CRISPR RNA and dynamically dependent on the conformational state of Cas13, as we show for several Cas13-family effectors encoded in one-vector and two-vector lentiviral systems. Moreover, host genes involved in viral processes and whose transcripts are intrinsically targeted by Cas13 contribute to constraining the lentiviral delivery and expression of Cas13. Our findings offer guidance for the appropriate use of lentiviral Cas13 systems and highlight the need for caution regarding intrinsic RNA targeting in Cas13-based applications.

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Fig. 1: Lentiviral defects of one-vector Cas13 systems.
Fig. 2: Lentiviral defects of two-vector Cas13 systems.
Fig. 3: Intrinsic RNA targets of different Cas13 effectors.
Fig. 4: Pre-crRNA cleavage activity underlies lentiviral defect of Cas13a.
Fig. 5: Endogenous RNA cleavage by Cas13a.
Fig. 6: Evaluation of Cas13 performance during high-throughput screening.

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Data availability

The data supporting the results in this study are available within the paper and its Supplementary information. The raw and analysed datasets generated in this study are available from the NCBI Gene Expression Omnibus database via the access code GSE202899. Source data are provided with this paper.

Code availability

The custom code for performing Lasso-based machine learning is available at https://github.com/cxlsky/Cas13-Lasso.

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Acknowledgements

We thank T. Xiao for technical advice and Y. Huang for sharing experimental material. This work was supported by the National Natural Science Foundation of China (31871344, 32071441), the Fundamental Research Funds for the Central Universities (N182005005, N2020001, N2220001), the 111 Project (B16009), the Key Laboratory of Bioresource Research and Development of Liaoning Province (2022JH13/10200026) and Liaoning Revitalization Talents Program (XLYC1807212) to T.F., and the research grant from the National Institutes of Health (R01HG010753) to W.L.

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Author notes

  1. These authors contributed equally: Zexu Li, Zihan Li, Xiaolong Cheng.

Authors and Affiliations

  1. Key Laboratory of Bioresource Research and Development of Liaoning Province, College of Life and Health Sciences, Northeastern University, Shenyang, China

    Zexu Li, Zihan Li, Shengnan Wang, Xiaofeng Wang, Shixin Ma, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Cheng Zhang & Teng Fei

  2. National Frontiers Science Center for Industrial Intelligence and Systems Optimization, Northeastern University, Shenyang, China

    Zexu Li, Zihan Li, Shengnan Wang, Xiaofeng Wang, Shixin Ma, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Cheng Zhang & Teng Fei

  3. Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang, China

    Zexu Li, Zihan Li, Shengnan Wang, Xiaofeng Wang, Shixin Ma, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Cheng Zhang & Teng Fei

  4. Center for Genetic Medicine Research, Children’s National Hospital, Washington, DC, USA

    Xiaolong Cheng, Lumen Chao & Wei Li

  5. Department of Genomics and Precision Medicine, George Washington University, Washington, DC, USA

    Xiaolong Cheng, Lumen Chao & Wei Li

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Contributions

T.F. and W.L. conceived the study and designed the research. Zexu Li and Zihan Li conducted most of the experiments. Zexu Li and X.C. conducted bioinformatics analysis. All the authors analysed the data. T.F. and W.L. wrote the paper with input from Zexu Li, Zihan Li and X.C., and help from all the other authors. T.F. and W.L. supervised the study.

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Correspondence to Wei Li or Teng Fei.

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W.L. is a paid consultant to Tavros Therapeutics, Inc. All other authors declare no competing interests.

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Nature Biomedical Engineering thanks Chunsheng Kang, Songying Ouyang and Li Yang for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Assessment of lentiviral defect for one-vector Cas13 systems.

a, Western blot of protein extracts from HEK293FT cells transiently expressing different Cas13 effectors in one-vector format. b, Immunocytochemistry of Cas13 proteins showing localization and expression. Scale bar, 20 µm. c-d, Surviving cell number after puromycin selection for T47D (c) or LNCaP (d) cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3, biological replicates. e, Cell viability analysis for HEK293FT cells with transient transfection of indicated one-vector lentiviral constructs, mean ± SEM with n = 3, biological replicates. Vector: lentiv2-w/o Cas9. f, Cell viability analysis for HEK293FT cells during lentivirus production by transient transfection of one-vector Cas13 along with packaging plasmids, mean ± SEM with n = 3, biological replicates. g-i, Assessment of surviving cell number (g), lentiviral RNA (h) and integrated lentiviral DNA (i) in A549 cells infected with indicated lentiviruses at low MOI. Data are mean ± SEM with n = 3, biological replicates (g), mean ± SD with n = 3, technical replicates (h-i).

Source data

Extended Data Fig. 2 Additional evaluation of lentiviral defect for one-vector Cas13 systems.

a-b, Time course examination of lentiviral RNA (a) or integrated DNA (b) post lentiviral infection in A549 cells, mean ± SD with n = 3, technical replicates. c-e, Assessment of surviving cell number (c), lentiviral RNA (d) and integrated lentiviral DNA (e) in A549 cells infected with indicated lentiviruses containing different crRNAs, mean ± SEM with n = 3. f, Evaluation of lentiviral defect for one-vector Cas13 systems under pHAGE-EF1α-puro plasmid backbone. Data are mean ± SEM with n = 3, biological replicates in cell survival assay, mean ± SD with n = 3, technical replicates in qPCR assays.

Source data

Extended Data Fig. 3 Evaluation of two-vector Cas13 systems for lentiviral defect.

a-c, Assessment of surviving cell number (a), lentiviral RNA (b) and integrated lentiviral DNA (c) in A549 cells infected with indicated lentiviruses at low MOI. d, Effect on cell survival after puromycin selection for A549 cells infected with high or low MOI of Cas13-only lentiviruses of two-vector system under pHAGE-EF1α-puro vector backbone, mean ± SEM with n = 3, biological replicates. e, Assessment of surviving cell number, lentiviral RNA and integrated lentiviral DNA levels using pHAGE-EF1α-puro vector backbone in A549 cells infected with indicated lentiviruses at high or low MOI. f, RNA levels in HEK293FT cells (left), HeLa cells (middle) and A549 cells (right) transiently transfected with pcDNA3.1-based Cas13 vectors by measuring NES element, mean ± SD with n = 3, technical replicates. g, Schematic of co-infection assay using indicated lentiviruses to evaluate lentiviral defect. Red arrowheads indicate detection region by qPCR at RNA and DNA levels. h-k, Assessment of surviving cell number (h) and lentiviral RNA levels using Cas13 (i), Cas13DR (j) or PuroR (k) elements in A549 cells co-infected with indicated lentiviruses. l, Integrated lentiviral DNA levels from genomic DNA of infected HeLa cells, U87 cells, SH-SY5Y cells and Neuro-2a cells by measuring NLS element, normalized to RPS28 or GAPDH (for SH-SY5Y), mean ± SD with n = 3, technical replicates. m-n, Cycloheximide (CHX) chase analysis for determining Cas13 protein stability in HEK293FT cells. Western blot (m) showing the protein levels at indicated time points and quantified band intensity was shown in (n).

Source data

Extended Data Fig. 4 Identification of intrinsic RNA targets of Cas13 by RIP-seq.

a, Schematic of RNA interactome and transcriptome analysis transiently transfected with indicated constructs in HEK293FT cells. Cas13b-crNT indicates a one-vector Cas13b system containing a non-targeting (NT) crRNA. Vector: lentiv2-w/o Cas9. b, The number of filtered strong RIP binding peaks (pileup > 15; fold enrichment > 4) in different samples. c, Loci and feature distribution of RIP-seq peaks for indicated samples. d, Top enriched motifs among RIP-seq peaks for indicated samples. e, Heatmap of genes associated with strong Cas13-bound peaks over the transcriptome. Vector: lentiv2-w/o Cas9. f, Top GO categories enriched among RIP-seq peak-associated genes across different samples.

Source data

Extended Data Fig. 5 Identification of intrinsic RNA targets of Cas13 by RNA-seq.

a, Heatmap of differentially expressed genes in Cas9- or Cas13-expressing HEK293FT cells. b, The number of differentially expressed genes that were up-regulated or down-regulated for indicated samples compared to vector control. c, Venn diagrams showing the overlaps of up- (left) or down-regulated (right) genes between Cas13a, Cas13b and Cas13d groups, versus vector. d-e, Top GO categories enriched among up- (d) or down-regulated (e) differentially expressed genes. f, Venn diagrams representing overlaps of up- (left) or down-regulated (right) genes in Cas13b or Cas13b-crNT groups, versus vector. g-h, RT-qPCR (g) and Western blot (h) validation for indicated gene expression in HEK293FT cells transiently transfected with lentiCRISPR v2-based Cas13b or Cas13b-crNT vectors, mean ± SD with n = 3, technical replicates for qPCR assay. i, Top 5 enriched GO categories of each Cas13-specific down- and up-regulated differentially expressed genes for Cas13a (top), Cas13b (middle) and Cas13d (bottom), versus vector.

Source data

Extended Data Fig. 6 Characterization of Cas13a variants.

a, Sequence alignment of catalytic domains for Cas13a derived from different bacteria strains. The conserved functional residues are shown in red star or by black arrow. b, Western blot of protein extracts from HEK293FT cells transiently transfected with indicated constructs. c, Coomassie blue staining showing purified Cas9 and Cas13a proteins. d, Cis RNA cleavage by indicated Cas13a variants during in vitro assay. Red arrow indicates the band position of on-target RNA or crRNA. e-f, Trans RNA cleavage by indicated Cas13a variants during in vitro assay. Results are shown by either fluorescence signal value (e) or direct visualization under blue light illuminator (f). a.u., arbitrary unit. mean ± SEM with n = 3, biological replicates. g, Pre-crRNA cleavage by indicated Cas13a variants during in vitro assay. Red arrows indicate band positions of intact and different cleavage patterns of RNA with structural schematic shown in right.

Source data

Extended Data Fig. 7 Profiling and validation of endogenous RNA targets by Cas13a variants.

a, The number of differentially expressed genes that were up-regulated or down-regulated among Cas13 variants compared to vector control. b-c, Top functional categories enriched among up- (b) or down-regulated (c) differentially expressed genes for indicated samples. d, RT-qPCR validation of indicated gene expression in HEK293FT cells transiently transfected with lentiCRISPR v2-based Cas13a and its variants, mean ± SD with n = 3, technical replicates. e, RNA expression change by RT-qPCR for indicated genes upon transient transfection with indicated constructs, mean ± SD with n = 3, technical replicates. f, Schematic for evaluating lentiviral capacity of HSPA6 knockdown by siRNA in HEK293FT cells. Lentiviruses (lentiCRISPR v2) produced in either control (siCtrl) or HSPA6 knockdown HEK293FT cells were used to infect A549 cells and lentiviral RNA or DNA level was determined. g, Assessment of HSPA6 knockdown efficiency in HEK293FT cells (left), lentiviral DNA (middle) and lentiviral RNA level (right) in A549 cells by measuring a region spanning NLS and FLAG tag in lentiCRISPR v2 vector, mean ± SD with n = 3, technical replicates. P values are determined via two-way ANOVA with Tukey’s multiple comparisons test or one-way ANOVA with Dunnett’s multiple comparisons test. h, RT-qPCR (left) and Western blot (right) analysis confirming the ectopic expression and knockdown of HSPA6 in HEK293FT cells. i, Integrated lentiviral DNA level of lentiCRISPR v2 vector in A549 cells by measuring NLS element after re-introduction of HSPA6 into HSPA6 knockdown HEK293FT cells by transient transfection, mean ± SD with n = 3, technical replicates.

Source data

Extended Data Fig. 8 Endogenous RNA targeting by Cas13.

a, RIP-qPCR validation of Cas13a binding sites. HEK293FT cells transiently expressing vector control (lenti-w/o Cas9) or Cas13a-Flag are analyzed. HA-IP serves as negative RIP control. GAPDH serves as negative site for Cas13a RIP enrichment. Values shown as mean ± SD with n = 3, technical replicates. *p < 0.001, two-way ANOVA with Sidak’s multiple comparisons test; ns means not significant. b-c, RT-qPCR (b) and Western blot (c) validation of indicated gene expression in HEK293FT cells transiently transfected with lentiCRISPR v2-based Cas13a and its variants. d, In vitro assay to assess the effect of Cas13a with differential conformation and complex constitution on endogenous RNA cleavage. e, Coomassie blue staining showing purified Cas9 and Cas13a/b/d proteins.

Source data

Extended Data Fig. 9 Cas13b/d-based cell fitness screens.

a, Schematic of computational pipeline to choose targeted RNA transcripts for Cas13b/d-based cell fitness screens. b, Statistics of sgRNA libraries for Cas13b/d-based screens. c, Scatter plot showing crRNA abundance change (log2 fold-change) in virus pool versus plasmid library for two biological replicates of either one-vector or two-vector Cas13d screens. R2, Pearson’s correlation coefficient. d, Scatter plot of β score (Day 33 versus Day 5) change for target transcripts between two biological replicates in Cas13d-null or Cas13d-expressing conditions of two-vector Cas13d screens. Red dots indicate core essential genes. e, The distribution of crRNA abundance across samples of different time points compared to plasmid pool for two-vector Cas13b screen. Boxes depict the upper and lower quartiles of the data, whiskers depict the maximum or minimum value within 1.5 times interquartile range above 75th or below 25th percentile, and black dots depict values that >1.5 times the interquartile range beyond either end of the box. f, The distribution of crRNA abundance (log2 fold-change) across samples of different time points compared to plasmid pool for two-vector Cas13b screens. g, Scatter plot of β score (Day 33 versus Day 5) change for target transcripts between two biological replicates in Cas13b-null or Cas13b-expressing conditions of two-vector Cas13b screens. Red dots indicate core essential genes.

Source data

Extended Data Fig. 10 Lasso model construction strategy and comparative summary of different types of Cas13 RNA targeting.

a, Early loss of crRNAs targeting non-essential genes in one-vector or two-vector Cas13d screens. Log2 fold-change (Day 5 vs. plasmid pool) < −0.5. b, Schematic of lasso-based machine learning model to determine crRNA-specific features underlying lentiviral defect. c, Comparison of features between different types of Cas13 activities.

Source data

Supplementary information

Supplementary Information

Supplementary discussion and references.

Reporting Summary

Supplementary dataset 1

Oligonucleotides, primers and crRNA sequences.

Supplementary Dataset 2

RIP-seq data.

Supplementary Dataset 3

RNA-seq data.

Supplementary Dataset 4

Cas13 libraries for high-throughput screening.

Supplementary Dataset 5

Cas13 screening data.

Supplementary Dataset 6

Unprocessed western blots and unprocessed gels.

Source data

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Li, Z., Li, Z., Cheng, X. et al. Intrinsic targeting of host RNA by Cas13 constrains its utility. Nat. Biomed. Eng 8, 177–192 (2024). https://doi.org/10.1038/s41551-023-01109-y

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