Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Abstract

Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.

Fig. 1. Generation and validation in vitro and in vivo of the enAsCas12a knock-in transgenic mouse.

A Diagram of the Rosa26-targeting construct for the genomic insertion of enAsCas12a, and the removal of the neo/stop cassette to enable constitutive expression. B NGS results showing the efficacy of constitutively expressed pre-crRNAs in MDFs (n = 3 each (WT and enAsCas12a^KI/KI MDF lines)). Cells were sequenced to assess gene editing at the target gene of the transduced pre-crRNAs. C NGS results showing the efficacy of the 4-tandem-guide construct (n = 3 each (WT and enAsCas12a^KI/KI MDF lines)). Cells were sequenced to assess gene editing at each of the target genes of the transduced 4-tandem-guide pre-crRNA array. D Schematic of the general process for haematopoietic reconstitution of WT mice with Eμ-Myc^T/+;enAsCas12a^KI/+ cells. E Combined survival curve of mice from two independent experiments that underwent haematopoietic reconstitution after transplantation with Eμ-Myc^T/+;enAsCas12a^KI/+ FLCs, transduced with either crTrp53 (n = 8) or crNTC (n = 10, with 5 mice having developed lymphoma). F NGS results for Trp53 editing from the tumourous tissue and concomitant lymphoma cell lines from mice (n = 7) reconstituted with the Eμ-Myc^T/+;enAsCas12a^KI/+;crTrp53 FLCs, demonstrating knockout efficacy. G Western blot validating TRP53 loss in independent Eμ-Myc^T/+;enAsCas12a^KI/+;crTrp53 cell lines (n = 5) derived from the splenic tissue of the reconstituted mice. Positive control cell line #19 was derived from a double-transgenic Eμ-Myc^T/+;enAsCas12a^KI/+ lymphoma-burdened mouse with a spontaneous Trp53 mutation. TRP53 stabilisation was induced via 24 h treatment with nutlin-3a (in the presence of QVD-O-Ph). HSP70 expression was used as a loading control. H, I NGS results showing the efficacy of in vivo gene editing of Trp53 (H) and Bim/Bcl2l11 (I) in the thymus and spleen of animals reconstituted with heterozygous enAsCas12a^KI/+ and homozygous enAsCas12a^KI/KI haematopoietic cells transduced with crNTC, crTrp53, or crBim (ex3) (n = 4 each). In all graphs, the mean is plotted, and error bars represent SD. Statistical analyses of NGS data can be found in Supplementary Data 4. Source data are provided as a Source Data file. Abbreviations: CAG cytomegalovirus enhancer, promoter of the chicken beta-actin gene, and splice acceptor of the rabbit beta-globin gene, NLS nuclear localisation signal, IRES = internal ribosome entry site, crRNA CRISPR RNA, MDF murine dermal fibroblast, FLCs foetal liver cells, WT wild-type, NTC non-targeting control.

Fig. 2. Applications of Cas12a ultra-compact, genome-wide, multiplexed murine-specific pre-crRNA libraries in vitro.

A Diagram of the design of the whole-genome screens in Eμ-Myc^T/+;enAsCas12a^KI/+ cells using the Menuetto (dual) and Scherzo (quad) pre-crRNA libraries. Eμ-Myc^T/+;enAsCas12a^KI/+ lymphoma cells were virally transduced (6 replicates) with each pre-crRNA library. After recovering, cells were treated (day 1) with either DMSO, nutlin-3a (2 μM), or S63845 (400 nM). On day 9, after multiple re-treatments (at the same concentrations), the cells were harvested for DNA. Indexing PCR was performed and then samples underwent NGS. B 4-way plot comparing the different arms of the screen samples for the Menuetto library. The y-axis compares S63845-treated samples with the input samples, and the x-axis compares the nutlin-3a-treated samples to the input samples. Significantly enriched hit genes are indicated in red, essential genes are indicated in orange, non-essential genes are indicated in dark grey, and other genes (genes not classified as either essential or non-essential) are indicated in light grey. A 4-way plot for the Scherzo library screen can be found in Supplementary Fig. 6C. Complete Eμ-Myc lymphoma-based screen analyses can be found in Supplementary Data 2. C Diagram of the design of the whole-genome drop-out screen in enAsCas12a^KI/KI iMDFs using the Menuetto pre-crRNA library. enAsCas12a^KI/KI MDFs were first immortalised (iMDFs) by viral transduction of SV40 large T antigen, and then iMDFs were virally transduced (3 replicates) with the Menuetto pre-crRNA library to achieve ~300x library coverage. Cells then underwent puromycin selection, before plating them for the screen. Cells were harvested on days 0, 4, 8, and 12 for DNA extraction. Indexing PCR and NGS were then performed. D 4-way plot comparing different arms of the iMDF drop-out screen using the Menuetto library. The y-axis displays the log2 fold change values for different genes at T2 vs T0, while the x-axis displays the log2 fold change values for different genes at T3 vs T0. Essential genes (indicated in orange) and are prominently lost over time. Significantly enriched tumour-suppressor genes are marked in red. Non-essential genes are indicated in dark grey, and other genes are indicated in light grey. Complete iMDF-based drop-out screen analyses can be found in Supplementary Data 3. Abbreviations: MDF murine dermal fibroblast, iMDF immortalised murine dermal fibroblast.

Fig. 3. Screening in vivo using the Menuetto library.

A Diagram of the design of the whole-genome in vivo screen using the Menuetto library and FLCs derived from Eμ-Myc^T/+;enAsCas12a^KI/+ crosses. B Survival curve of mice transplanted with Eμ-Myc^T/+;enAsCas12a^KI/+ FLCs for in vivo screening with the Menuetto pre-crRNA library. crNTC mice remain mostly alive after 95 days, with only one mouse succumbing to pre-B/B cell lymphoma (n = 1/6). Two crNTC control mice have been excluded from the curve (original cohort n = 8) as their death was determined to have a cause other than pre-B/B cell lymphoma (via immunophenotyping). crTrp53 control mice (n = 5/5) reached ethical endpoint with a median latency of 25 days. Menuetto library mice (n = 17/21) have reached ethical endpoint with a median latency of 48 days. Mantel-Cox log-rank test comparisons: crNTC vs Menuetto p = 0.0174, crNTC vs crTrp53 p = 0.0008, crTrp53 vs Menuetto p < 0.0001. C Immunophenotyping results from the tumours of the 17 Menuetto library mice that have so far succumbed to lymphoma. D Graph of highly enriched pre-crRNAs in the haematopoietic tissue tumours of the 17 mice obtained so far from the in vivo Menuetto library screen. Trp53-targeting pre-crRNAs were the most common, and likely causative even in the instances where multiple, roughly equally represented pre-crRNAs were detected (Trp53 + other; n = 6). Source data are provided as a Source Data file. Abbreviations: crRNA CRISPR RNA, FLCs foetal liver cells, WT wild-type, NTC non-targeting control, NGS next-generation sequencing.

Fig. 4. Multiplexing of enAsCas12a and dCas9-SAM in OT-I T cells and MDFs.

A Quantification of FACS analysis of enAsCas12a^KI/+;dCas9-SAM^KI/+;OT-I^T/+ T cells transduced with an sgCd19 (BFP-tagged) for dCas9-SAM-mediated gene expression and a crTrp53 (GFP-tagged) for Cas12a-mediated gene editing (n = 3 independent transductions). ~6% of cells from the sample transduced with both constructs were both GFP- and BFP-positive. B Quantification of FACS analysis of the T cells transduced with lentiviral vectors expressing crTrp53 and sgCd19, or crTrp53 and sgNTC (n = 3 independent transductions each). sgCd19 was able to induce a statistically significant increase in CD19 expression in the double-transduced cells (Student’s t-test (two-tailed), t = 125.2, df = 4, p < 0.0001). C NGS data measuring Trp53 gene editing from the CD19+ populations of T cells transduced with sgCd19/crTrp53, sorted depending on their GFP status (n = 1). D Quantification of CD19 mean fluorescence intensity (MFI) in multiplexed enAsCas12a^KI/+;dCas9^KI/+ MDFs, transduced with a single vector co-expressing sgCd19 and either crTrp53 or crBax/crBak (n = 3 independent transductions). Cells were gated on mCherry, GFP, and BFP expression. Significant upregulation of CD19 was observed in MDFs transduced with the pMS2-BFP U6_sgCd19 H1_crTrp53 vector (Kruskal-Wallis test with multiple comparisons to the empty vector control, p = 0.0225). E Western blots demonstrating TRP53, BAX, and BAK loss in the multiplexed enAsCas12a^KI/+;dCas9^KI/+ MDFs (n = 3) or those transduced with the empty vector (n = 1). F Quantification of the western blots shown in (E). Clear loss of both TRP53 and BAX was observed in all samples, but BAK loss was less efficient. G NGS results from enAsCas12a^KI/+;dCas9^KI/+ MDFs (n = 3 per transduction), transduced with the pMS2-BFP empty vector, or with pMS2-BFP containing either sgCd19/Trp53 or sgCd19/crBax/crBak. Cells were sorted based on BFP/CD19 status prior to DNA extraction. Target gene editing was higher in cells that were BFP + CD19+ than in those that were BFP + CD19-. In each graph, the means are plotted, and the error bars represent SD. p < 0.05 = *, p < 0.0001 = ****. Statistical analyses of NGS data can be found in Supplementary Data 4. Source data are provided as a Source Data file. Abbreviations: MDF murine dermal fibroblast.