Advancing the genetic engineering toolbox by combining AsCas12a knock-in 1 mice with ultra-compact screening

Abstract


Introduction
CRISPR-Cas9, the first developed CRISPR-Cas-based gene-editing variant, has found near unparalleled utility in biological and medical research.A particular strength of using CRISPR-Cas9 is the ability to undertake rapid, targeted genetic screens to identify, for example, genes involved in tumourigenesis or clinical drug resistance [1,2].Cas12a (Cpf1) is an RNA-guided endonuclease that distinguishes itself from Cas9 by its short CRISPR RNAs (crRNAs), intrinsic RNase activity, and different protospacer adjacent motif (PAM) requirements [3].RNase activity is necessary for Cas12a to extract mature crRNAs from precursor transcripts (pre-crRNA arrays) by recognition of direct repeat (DR) hairpins upstream (5') of the crRNA targeting sequence.Concatenating multiple guide RNAs within a single pre-crRNA array is therefore possible, and enables combinatorial targeting of one or multiple genes from a compact, easily-clonable, RNA Pol-III expression cassette [4,5].The gene-editing effectiveness of Cas12 has been improved by the engineering of enhanced Acidaminococcus sp.-derived Cas12a (enAsCas12a) [6], but this was previously only applicable in mammalian cells in vitro.
To extend the applications of Cas12a-mediated gene-editing, we have generated an enAsCas12a knock-in transgenic mouse and demonstrated its efficacy when targeting genes individually or simultaneously in vitro in primary cells and cell lines, as well as in vivo via haematopoietic reconstitution.We have furthermore developed two murinespecific, ultra-compact, pre-crRNA libraries, enabling highly effective genome-scale CRISPR-Cas12a knockout screens in cells derived from this model.

Generation and characterisation of the enAsCas12a mouse
To introduce Cas12a in vivo, we obtained the E174R/S542R/K548R-substitution variant of Acidaminococcus sp.Cas12a (enAsCas12a; derived form an unclassified Acidaminococcus strain (BV3L6)) [6], which has previously been utilised in functional genomic screening [7,8].The enAsCas12a open reading frame was further modified to contain additional nuclear localisation sequences, which can improve enzyme functionality [9].The enAsCas12a cDNA was then cloned into the Cre-recombinaseinducible expression cassette of a previously described mouse Rosa26-targeting construct [10], further modified to include an IRES-mCherry marker (instead of IRES-GFP), and enAsCas12a knock-in (enAsCas12a KI ) mice were then generated by pronuclear microinjection of this construct into C57BL/6 one-cell stage embryos.We confirmed enAsCas12a insertion by long-range PCR (Fig. S1A).Once generated, homozygous enAsCas12a KI/KI mice were crossed with CMV-Cre mice to remove the loxP-flanked neo/stop cassette and allow constitutive, whole-body enAsCas12a expression (Fig. 1A, S1B).This was demonstrated by IRES-mCherry expression in haematopoietic cells/tissues via FACS; almost 100% of peripheral blood cells were mCherry+, and ~80% of haematopoietic organ cells (thymus, bone marrow, spleen, and lymph nodes) had detectable marker fluorescence (Fig. S1C).To assess enAsCas12a expression-related toxicity, we analysed haematopoietic compartment cell populations, specifically B, T, and myeloid cell types.No changes were observed compared to wildtype controls, suggesting constitutive enAsCas12a expression was well-tolerated (Fig. S2A-D).Furthermore, no health issues were observed in transgenic mice aged up to 250 days (data not shown).Together, these data suggest our enAsCas12a mouse model is healthy, and has consistent whole-body expression of the enAsCas12a transgene.

Validation of the enAsCas12a gene-editing efficacy in vitro
To evaluate enAsCas12a activity, murine dermal fibroblasts (MDFs) from enAsCas12a KI   or WT mice were isolated and transduced with lentiviral vectors for constitutive expression of individual crRNAs targeting Trp53 (exon 4) or Bim/Bcl2l11 (exon 2 or 3) (Table S1).Target editing efficiency in transduced enAsCas12a KI/KI MDFs was ~100% for each locus, as determined by next generation sequencing (NGS) (Fig. 1B).TRP53 loss was also confirmed via western blotting, with no protein observed even 6 h after treatment with nutlin-3a (MDM2 inhibitor, which leads to indirect TRP53 activation [11]) (Fig S1D).We next adapted our previously described inducible guide RNA expression platform [12], allowing for doxycycline (dox)-mediated temporal control of pre-crRNA activity, for use in combination with our enAsCas12a-engineered mouse model.After targeting Trp53 or Bim/Bcl2l11 in enAsCas12a KI/KI MDFs, NGS revealed up to 100% target gene-editing efficiency 24 h post-dox treatment (Fig. S3A, B).At baseline (predox treatment), ~70-80% of alleles showed signs of editing, representing background leakiness of the inducible promoter.We then extended our investigations to examine the potential for multiplexed geneediting via enAsCas12a.A 4-tandem-guide construct was designed for simultaneously targeting Trp53, Bim/Bcl2l11, Puma/Bbc3, and Noxa/Pmaip1 from a single pre-crRNA expression cassette, with the guides separated by unique DRs on their 5' end (Fig. 1C).Following lentiviral delivery of this construct, we observed 100% editing efficiency for each targeted gene in enAsCas12a KI MDFs, and none in WT MDFs (Fig. 1D).NGS also demonstrated 100% gene-editing efficiency for each gene in the dox-treated MDFs using the inducible 4-tandem guide construct.However, as for the inducible single gene pre-crRNAs, leakiness was also observed in pre-dox treated enAsCas12a KI/KI MDFs (Fig. S3C).To expand our assessment of enAsCas12a activity to other cell types and biological contexts, homozygous enAsCas12a KI/KI female mice were crossed with Eμ-Myc T/+ males, and we generated Eμ-Myc T/+ ;enAsCas12a KI/+ B lymphoma cell lines from mice that developed MYC-driven lymphoma [13,14].We then tested all our constitutive and inducible guides, following lentiviral delivery, in three independent Eμ-Myc T/+ ;enAsCas12a KI/+ -derived cell lines, and obtained ~50% gene-editing efficiency for both Trp53 and Bim/Bcl2l11 when individually targeted (Fig. S4A, B).Multiplex targeting was more variable, resulting in ~15% (for the constitutive construct) or ~35% (for the inducible construct) gene-editing efficiency for Trp53, ~10% (constitutive) or ~20% (inducible) for Bim/Bcl2l11, and less for both Puma/Bbc3 and Noxa/Pmaip1 (Fig. S4C,   D).In addition, there was less leakage as measured by indel/frameshift mutations predox treatment in Eμ-Myc T/+ ;enAsCas12a KI/+ cells than in the enAsCas12a KI/KI MDFs.This suggests that the enAsCas12a system may vary in efficacy based on cell type, or that the presumably higher expression of enAsCas12a in homozygous enAsCas12a KI/KI MDFs results in more robust activity.Collectively, these data demonstrate our enAsCas12a model is capable of high efficiency single and multiplex gene-editing in vitro, using either constitutive or inducible pre-crRNA vectors.

Validation of the enAsCas12a gene-editing efficacy in vivo
Having established the efficiency of our enAsCas12a system in vitro in both primary and transformed cell lines, our next aim was to evaluate in vivo functionality.To do so, we performed haematopoietic reconstitutions, first transducing Eμ-Myc T/+ ;enAsCas12a KI/+ foetal liver cells (FLCs; obtained at embryonic day 14) with a constitutive Trp53targeting crRNA construct or empty vector control ex vivo, then transplanting the transduced FLCs into lethally-irradiated C57BL/6 recipient mice before monitoring for lymphoma development (Fig. 2A).All mice transplanted with Trp53-targeting pre-crRNA-transduced FLCs developed lymphoma by 28 days post-transplantation, (consistent with the latency of reconstituted Eμ-Myc T/+ ;Trp53 KO mice [12,15]) (Fig. 2B).FACS analyses of the haematopoietic tissues revealed the tumours were primarily immature pro-/pre-B cells (B220 + IgM -IgD -) (Fig. 2C).In cell lines derived from the haematopoietic tissues of these mice, successful enAsCas12a-mediated Trp53 knockout was confirmed by NGS and western blotting analysis (Fig. 2D, E).Collectively, these findings demonstrate that our enAsCas12a system is effective for in vivo experimentation.Creation and functional assessment of the Menuetto and Scherzo libraries for whole-genome knockout screening in enAsCas12a KI/+ mouse-derived cells While several genome-scale crRNA expression libraries compatible with Cas12a have been described for use in human cells (Humagne [7], a "dual" library where there are 4 unique crRNAs per gene across 2 constructs, and Inzolia [8], a "quad" library where 4 unique crRNAs per gene are within a single construct), there are no publicly-disclosed equivalents for screening in murine cells.Therefore, we developed two compact, genome-wide, murine-specific pre-crRNA libraries: Menuetto (dual) and Scherzo (quad).
The Menuetto library contains 43,920 constructs with pre-crRNAs targeting 21,743 genes, along with 500 non-targeting controls (NTCs), while the Scherzo library contains 22,839 constructs with pre-crRNAs targeting 21,721 genes plus 500 NTCs (Supplementary File 1).To demonstrate the utility of the Menuetto and Scherzo libraries, we performed genetic screens using one of our aforementioned Eμ-Myc T/+ ;enAsCas12a KI/+ cell lines (#20).Six replicate transductions were performed for each library before cells were treated with either DMSO, nutlin-3a (2 μM, retreated 4 times; an MDM2 inhibitor, which leads to indirect TRP53 activation [11]), or S63845 (400 nM, retreated 2 times; an inhibitor of the pro-survival BCL-2 family protein MCL-1 [16]) (Fig. 3A).Drug concentrations close to the IC 50 values were chosen, as determined via preliminary viability assays (Fig. S5).Once the cells recovered from the multiple treatments, DNA was isolated and NGS was performed to identify enriched crRNAs.Analyses of both the Menuetto (Fig. 3B) and Scherzo (Fig. 3C) library screens revealed similar results: strong enrichment of Trp53targeting pre-crRNAs when comparing nutlin-3a-treated cells to the input samples, and strong enrichment of Bax-targeting crRNAs when comparing S63845-treated cells to the input samples.This is in line with expectations, as we have previously identified and validated Trp53 and Bax as resistance factors to nutlin-3a-and S63845-mediated killing, respectively, after conducting whole-genome genetic screens using CRISPR-Cas9 in Eμ-Myc cells [1,2,17].By contrast, the DMSO-treated samples showed no standout guide enrichment for either library (complete analyses of the screens can be found in Supplementary File 2).These data suggest the Menuetto and Scherzo libraries perform well as compact CRISPR-Cas12a whole-genome knockout libraries for use in murine enAsCas12a transgenic cells.

Combinatorial gene modification: Cas12a-mediated gene knockout with dCas9a-SAM-induced gene expression
Next, we wanted to explore the potential for our enAsCas12a mouse to be used for inter-Cas multiplexing applications.In addition to facilitating multiplexed gene knockout through pre-crRNA processing, Cas12a can also be multiplexed with orthogonal Cas molecules, such as a dCas9a used for CRISPR activation (CRISPRa), as their PAM/targeting requirements and guide RNA scaffolds specificities are distinct.To perform such an experiment, we first crossed enAsCas12a mice to OT-I Tg mice (OT-I; in which the T cell receptor is engineered to recognise the immunogen, ovalbumin (OVA) [18]), and the resulting progeny were crossed with our previously described CRISPRa dCas9a-SAM mice [19] to generate enAsCas12a KI/+ ;dCas9a-SAM KI/+ ;OT-I T/+ mice.Splenocytes from these mice were stimulated with OVA peptide and transduced with either a Cd19-targeting sgRNA (BFP co-expression), a Trp53-targeting crRNA (exon 4targeting; GFP co-expression), or both in combination.Approximately 6% of cells were successfully transduced with both constructs (Fig. 4A, S6A), of which ~55% expressed CD19, demonstrating successful dCas9a-SAM-mediated Cd19 activation (Fig. 4B, S6B).We next isolated GFP-negative and GFP-positive cells from the CD19+ population, and used NGS to assess the level of Trp53 gene-editing.As expected, we observed no genetic perturbations of Trp53 in CD19+GFP-cells, while Trp53 was edited with ~50% efficiency in CD19+GFP+ cells (Fig. 4C, S6C).The success of this enAsCas12a/dCas9a-SAM multiplexing led us to question whether the technique could be performed using a single transduction.To test this, we cloned either the Trp53-targeting (exon 4-targeting) crRNA or an array containing both Baxand Bak-targeting crRNAs into the same vector as the Cd19-targeting sgRNA.Then, we transduced these vectors into enAsCas12a KI/+ ;dCas9a KI/+ MDFs (or the empty vector as a control), and examined gene activation efficacy by flow cytometry 8 days posttransduction.FACS analysis first demonstrated that the majority of our transduced enAsCas12a KI/+ ;dCas9a KI/+ MDFs were triple-fluorescent (mCherry co-expression from enAsCas12a, GFP co-expression from dCas9a-SAM, and BFP co-expression from the crRNA/sgRNA vector), a useful property for multiplexing applications (Fig. S6D).In those triple-fluorescent cells, we were then also able to observe strong CD19 induction relative to those MDFs transduced with the empty vector (Fig. 4D, S6E).We then examined the efficacy of our gene-knockout via western blotting.For each of the enAsCas12a KI/+ ;dCas9a KI/+ MDFs transduced with the sgCd19/crTrp53 vector, we observed clear loss of TRP53 expression (Fig. 4E, F).Similarly, for enAsCas12a KI/+ ;dCas9a KI/+ MDFs transduced with the sgCd19/crBax/crBak vector, we observed clear loss of BAX, and strong (but incomplete) loss of BAK (Fig. 4E, F).Together, these data demonstrate that multiplexing enAsCas12a with dCas9a-SAM is a viable strategy for simultaneous knockout and activation of distinct target genes.

Discussion
In this study, we describe the generation of a murine model containing a constitutivelyexpressed enhanced Acidaminococcus sp.-derived Cas12a construct (enAsCas12a).We were able to validate that constitutive enAsCas12a expression is well-tolerated, particularly with respect to the haematopoietic system.Furthermore, enAsCas12a expression is present in all surveyed tissues through functional assays and detection of a linked mCherry reporter gene.Efficacy of enAsCas12a was determined both in vitrousing MDF and Eμ-Myc pre-B/B cell lymphoma cell lines with constitutively and inducibly expressed pre-crRNAs -and in vivo -using mice haematopoietically reconstituted with Eμ-Myc T/+ ;enAsCas12a KI/+ foetal liver cells transduced with constitutively expressed pre-crRNAs.Differences in the efficacy of gene perturbation activity across assayed contexts could likely be explained by enAsCas12a dosage, either due to the genetic backgrounds (homozygous vs heterozygous), cell typedependent differences with the CAG promoter driving enAsCas12a, or some level of selection favouring the edited cells (e.g.Trp53 loss inhibiting cell death).We further designed and synthesised two whole-genome, multiplexed, mouse-specific Cas12a crRNA expression libraries: Menuetto (dual) and Scherzo (quad), and demonstrated their utility for in vitro screens using Eμ-Myc lymphoma cells.Finally, we crossed our enAsCas12a mouse with our previously generated dCas9a-SAM mouse [19], and demonstrated the capability for multiplexed experiments with our models, simultaneously utilising both Cas molecules in primary T cells and MDFs to activate CD19 expression and mutate Trp53 or Bax/Bak.Notably, these experiments also demonstrated the viability of our enAsCas12a system as being functional in a heterozygous context, meaning it could easily be crossed to disease models to enable broad application amongst the scientific community.Multiplexing experiments are an emerging area of utility for CRISPR technologies, which exploit the specificity of different Cas molecules to their particular pre-crRNAs/sgRNAs.To utilise this capability to its maximum potential, we designed our enAsCas12a mouse with an mCherry marker, making it compatible with our previously generated dCas9a-SAM mouse, which has a GFP marker [19].This design choice should improve quality-of-life for the end users, permitting easy detection and/or enrichment of Cas-/construct-positive cells when needed.Combined with, for example, a BFP-tagged sgRNA/pre-crRNA expression vector, we have demonstrated that one can easily obtain triple-fluorescent (or potentially more) cells with significant utility in FACS and imaging applications.Due to the RNase capability of Cas12a, multiplexing is also simplified at the level of guides, as multiple crRNAs can be generated from a single RNA molecule and a single promoter.We tested both inducible and constitutively-expressed versions of individual pre-crRNAs and a 4-tandem-guide construct (targeting Trp53, Bim/Bcl2l11, Puma/Bbc3, and Noxa/Pmaip1 in parallel) in vitro using primary enAsCas12a KI/KI MDFs and Eμ-Myc T/+ ;enAsCas12a KI/+ lymphoma cells.We saw no loss of gene-editing efficacy in primary MDFs when comparing the constitutive and inducible individual and 4-tandemguide configurations, but enAsCas12a activity was observed without dox-treatment for both inducible configurations in enAsCas12a KI/KI MDFs.This leakiness was absent in Eμ-Myc T/+ ;enAsCas12a KI/+ lymphoma cells transduced with the 4-tandem-guide construct, but there was a marked drop in gene-editing efficiency for those pre-crRNAs distal to their promoter.Previous studies have also reported variable efficiencies in either crRNA cleavage or concomitant gene-editing when using multiplexed Cas12a crRNAs, in various species ranging from bacteria and yeast to human cells [5,[20][21][22].As we did not observe a reduction in gene-editing efficiency using the 4-tandem-guide constructs in enAsCas12a KI/KI MDFs, but lower Bak (positioned 3' of the Bax-targeting guide in the Bax/Bak crRNA array) gene-editing in the enAsCas12a KI/+ ;dCas9a KI/+ MDFs, this suggests these differences in efficiency are likely dependent on differences in Cas12a expression level.Multiplexing can also refer to multiple guides targeting a single gene, such as has been the case in the previously generated human-specific Cas12a crRNA expression libraries; Humagne [7] and Inzolia [8].Humagne uses 2 constructs per gene, each with 2 unique pre-crRNAs (dual), while Inzolia uses only one construct per gene with 4 unique pre-crRNAs (quad).We have taken a similar approach in designing two whole-genome, mouse-specific Cas12a crRNA libraries; Menuetto (dual) and Scherzo (quad).There are advantages and disadvantages to each approach.For example, our dual-style Menuetto library potentially holds more analytical power, as multiple crRNA constructs can be detected per gene, which is necessary for some popular CRISPR screen analysis methods (e.g.MAGeCK [23]) to assign statistical significance.Alternatively, our quad Scherzo library is approximately half the size of the dual Menuetto library, which is advantageous for in vivo applications due to the maximisation of gene coverage across a restricted number of guide-expressing cells.Regardless, our screening approach suggests both the Menuetto and Scherzo libraries are highly effective.We obtained a number of expected hits, including Trp53, Bax, Puma/Bbc3, and Bim/Bcl2l11, all of which are well-characterised as being necessary for S63845-or nutlin-3a-mediated apoptosis in MYC lymphomas [1,17,24,25].Other strong hits, such as Naa10, are less well characterised, but have been reported elsewhere as necessary for TRP53mediated apoptosis, as well as having been a hit in a similar CRISPR-Cas9 screen performed by our group [2,26].We will make the Menuetto and Scherzo libraries available in the near future to the wider scientific community and look forward to their discoveries.In conclusion, our resultsdemonstrate the power of Cas12a as an extension of our gene-editing toolbox for engineering sophisticated pre-clinical models and interrogating complex biological pathways.

Small-scale Cas12a pre-crRNA design and construct cloning
The Cas12a individual pre-crRNAs and 4-tandem-guide array targeting Trp53, Bim/Bcl2l11, Bbc3/Puma, and Pmaip1/Noxa were designed using Benchling (sequences given in Table S1) and ordered from Integrated DNA Technologies (IDT).Individual pre-crRNAs were synthesized with the same DR (5'-TAATTTCTACTCTTGTAGAT-3') at the 5' end, which is the sequence recognized by Cas12a, as well as with 4 bp overhangs at the 5' end for the complementary (5'-TCCC-3') sequence and reverse complementary (5'-AAAC-3') sequences, before being cloned into the BsmBI site (vectors had been modified to contain this site) of either the constitutive lentiviral vector FUGW (Addgene #14883) or the doxycycline-inducible vector FgH1tUTG (Addgene #70183).The 4-tandem-guide array was similarly cloned into the BsmBI site of either FUGW or FgH1tUTG.The dCas9a-SAM-compatible Cd19-targeting sgRNA vector was generated by first modifying the lenti sgRNA(MS2)_puro optimized backbone (Addgene #73797) to remove the puromycin selection sequence and instead incorporate a BFP marker.The Cd19 sgRNA sequence (Table S1) was then cloned into the BsmBI site of the modified vector.The sequences encoding the H1 promoter and crRNA targeting Trp53 or the crRNA array targeting Bax/Bak were cloned into the BamHI site of the modified vector.

Cell sorting and doxycycline-induction
After 2 days of recovery post-transduction, MDFs and Eμ-Myc T/+ ;enAsCas12a KI/+ cells were sorted using an Aria III (BD Biosciences) to acquire only cells with enAsCas12a-IRES-mCherry and pre-crRNA-GFP expression.Cells with inducible constructs were treated with doxycycline (10 μg/mL) for 24 h to induce pre-crRNA expression as per established protocols [12].

DNA extraction and next generation sequencing
In all instances, genomic DNA (gDNA) was extracted from cells using DNeasy Blood & Tissue kit (QIAGEN #69506).DNA samples were prepared for NGS similarly to previous descriptions [12].In short, primers that flank one of the pre-crRNA sequences in the 4-tandem-guides construct were designed with 5' overhangs and ordered from IDT (full primer sequences in Table S2).To amplify each gene region, an initial PCR was performed using gDNA (100 ng) with 1x GoTaq Green Master Mix (Promega #M7123), and 0.5 μM of each primer.Cycling conditions were 18 cycles of 95°C for 2 m, 60°C for 30 s, 70°C for 30 s.To index the samples for sequencing, a second PCR reaction was then performed using the product from the first PCR (1 μL) with 1x GoTaq Green and 0.5 μM of each primer (indexing sequences in Table S3).The cycling conditions were 24 cycles of 95°C for 2 m, 60°C for 30 s, and 70°C for 30 s. From each amplified, indexed PCR product, 5 μL was pooled and purified using 1.0x Ampure Beads (Beckman Coulter #A63880), and the pooled samples were sequenced using a MiSeq (Illumina).Indels were quantified using the CRISPR indel calculator (http://crisprindelcalc.net).

Western blot analyses
Eμ-Myc T/+ ;enAsCas12a KI/+ ;crTrp53 cell lines derived from lymphoma-burdened reconstituted mice (#205 #207) were treated with the pan-Caspase inhibitor QVD-O-Ph (25 μM; MedChemExpress #HY-12305) for 15 min to inhibit cell demolition, before being treated with nutlin-3a (10 μM; MedChemExpress #HY-10029) for 24 h to induce TRP53 activation (control cells were treated with DMSO).MDF samples (except those in the multiplexing experiments) were treated with nutlin-3a (10 μM) for 6 h with no QVD-O-Ph, but otherwise prepared the same way.Cell pellets were collected and resuspended in RIPA lysis buffer (NaCl (300 mM), SDS (0.2%), Triton X-100 (2%), sodium deoxycholate (1%), Tris HCl (100 mM, pH 8.0)) with protease inhibitor (Roche #11836145001), then incubated on ice for 30 m. Protein-containing supernatant was collected after centrifugation at 13000xg for 10 m at 4°C.To quantify protein content, a BCA assay (Thermo Fisher Scientific #23225) was performed, except for the multiplexing experiments where equal cell numbers were used.Then, 25 μg of protein (or all of the sample for the MDF multiplexing experiment across 2 gels) was loaded into a NuPAGE 4~12% Bis-Tris 1.5 mm gel (Invitrogen #NP0335) and gel electrophoresis performed.Protein was transferred onto a nitrocellulose membrane (Invitrogen #IB23002) according to the manufacturer's instructions, and the membrane blocked with 5% skim milk powder dissolved in PBS-T (1x PBS with 0.1% Tween-20 (Sigma-Aldrich #P1379)) for ~1 h at room temperature, and then incubated in primary antibody against P53 (1:2000; Novocastra #NCL-p53-CM5p), β-ACTIN (1:2000; Sigma #A2228), BAX (1:2000; Sigma Aldrich #B9054), BAK (1:2000; Sigma Aldrich #5897), or HSP70 (1:10,000; gift from Dr R Anderson, ONJCRI) (dissolved in PBS-T) at 4°C overnight, with agitation.The next day, the membrane was washed ~3 times with PBS-T before incubation with HRP-conjugated anti-rabbit (Southern Biotech #4010-05) or anti-mouse (Southern Biotech #1010-05) secondary antibody.The protein bands were visualised by adding Immobilon Forte Western HRP substrate (Millipore #WBLUF0100) on a ChemiDoc XRS+ (BioRad).crRNA design for the Menuetto library (dual pre-crRNA library) For each mouse protein-coding gene found in Ensembl Release 102, we designed 2 pairs of 23mer spacer sequences for the enAsCas12a nuclease based on the enAsCas12a design rules implemented in the crisprVerse [30].First, we filtered out spacer sequences with at least one of the following characteristics: contains a poly-T stretch, has GC content below 20% or above 80%, or contains a recognition site for the restriction enzymes EcoRI and KpnI used for lentiviral cloning.Spacer sequences were then selected to (1) minimize the number of putative off-targets located in other coding sequences, (2) optimize on-target activity using the enPAM+GB prediction algorithm described in [7], and (3) target the canonical isoform as defined by Ensembl.We required a minimal distance of 25 nucleotides between spacer sequences within a pair to avoid competing of nuclease occupancy, and required at least 50 nucleotides between pairs as well.When possible, spacers for a given gene were chosen across different exons to increase the probability of having a functional knockout.Spacers located in known Pfam domains, as well as in the first 85% of the CDS region, were prioritized.Finally, to avoid the unintended deletion of functional non-coding elements, we constrained each pair of spacers to regions that do not overlap known non-coding elements described in Ensembl Release 102 (miRNAs, tRNAs, lncRNAs, rRNAs, snRNAs, and snoRNAs).We also included 500 pairs of non-targeting controls (NTCs).The final library, named Menuetto, contains a total of 46,242 pairs of spacer sequences (full Menuetto library information can be found in Supplementary File 1).Pre-crRNAs for the Menuetto library (and Scherzo library, see below) were synthesized and cloned (Cellecta) into a vector derived from the pLKO.1 vector (Addgene #10878) with pre-crRNAs under control of the hU6 promoter and BFP under control of the EF-1α promoter.crRNA design for the Scherzo library (quad pre-cRNA library) The design of the 4 spacers per gene for the quad pre-cRNA library, named Scherzo, follows the design of the Menuetto library.The 4 spacers per gene are also constrained to a region that does not overlap known non-coding elements.As a result, there is a small number of genes for which it is not possible to design one quad array that targets all isoforms of a given gene without overlapping non-coding elements.For such cases, we designed an additional quad array to target the remaining isoforms.The final library contains a total of 22,839 quad arrays, including 500 NTC quad arrays (full Scherzo library information can be found in Supplementary File 1).

Genome-wide Menuetto and Scherzo crRNA library sequencing
Virus production and Eμ-Myc T/+ ;enAsCas12a KI/+ cell transduction was performed as described above, with 10 μg library DNA used for each of the six independent transductions undertaken (300,000 cells per transduction) for each library.Eμ-Myc T/+ ;enAsCas12a KI/+ cells used for screening were not sorted/selected.After recovering from transduction, cell replicates were expanded into T75 flasks (Corning #430641), and treated with either DMSO, Nutlin-3a (2 μM, retreated 4 times; an MDM2 inhibitor, which leads to indirect TRP53 activation), or S63845 (400 nM, retreated 2 times; an MCL-1 inhibitor).Starting drug concentrations were chosen to be around the IC 50 value, as determined by viability assays performed as previously described [2] (Fig. S5).The first drug treatments occurred ~7 days post-transduction.After recovering from the final treatment, pellets of 4 million cells were collected, washed with 1x PBS, and the DNA extracted as above.crRNAs were then amplified from 250 ng of DNA using GoTaq Green according to the manufacturer's instructions, and the following PCR protocol: 3 m at 95°C, [15 s at 95°C, 30 s at 60°C, 30 s at 72°C repeated 35 times], and 7 m at 72°C.The primers used for amplification and indexation of the pre-crRNAs are given in Table S4.PCRs were performed in duplicate for each sample, with each library indexed separately using the same primer combinations.Products were pooled for each library separately, then cleaned up using Ampure XP beads (Beckman Coulter #A63881) and sequenced on a NextSeq 2000 (Illumina) according to the manufacturer's instructions.

Statistical analyses for the Menuetto and Scherzo screens
For both screens, reads were mapped to the libraries using MAGeCK v0.5.9 [23] to generate the raw count data.Raw count data were then stored in a standard Bioconductor SummarizedExperiment object [31] and normalized for sequencing depth.We performed a differential abundance analysis for each pair or quad of pre-crRNAs separately using the popular limma-voom approach [32].Specifically, we fitted a linear model to the log-CPM values for each pre-crRNA array, using voom-derived observation and quality weights.We performed robust empirical Bayes shrinkage to obtain shrunken variance estimates for each pre-crRNA array, and we used moderated F-tests to compute p-values for each of the two-group comparisons of interest.For the Menuetto screen, we obtained gene-level statistics by aggregating statistics per gene from the each gene-specific pre-crRNA pair.In particular, we used the "fry" geneset enrichment analysis method implemented in limma, and considered the two pairs targeting a given gene as a "gene set".This allows the detection of genes that are consistently enriched or depleted for the two pre-crRNA pairs.We applied the Benjamini-Hochberg procedure to obtain an FDR-corrected p-value for each gene.Essential/non-essential genes were determined as previously described [33].Hits were selected by using an FDR threshold of 20%.For the Scherzo screen, the quad array p-values were corrected for multiple comparisons using the Benjamini-Hochberg procedure.Hits were selected by using an FDR threshold of 5%.Primary mouse T cell culture, transductions, and cell sorting Virus production was performed as described above, with 60 μg DNA used in the transduction (~100x10 6 splenocytes).Non-treated 6-well plates were coated with retronectin (48 μg/well) overnight at 4 o C, washed with 1x PBS and blocked with 2% BSA for 30 m, before the addition of filtered viral supernatant.For multiplexing, each virus was generated independently before supernatants were combined (1:1).Virus was bound to retronectin-coated plates by centrifugation (3500 rpm, 2 h, 32 o C), and the supernatant removed.enAsCas12a KI/+ ;dCas9a-SAM KI/+ ;OT-I T/+ splenocytes were resuspended at ~1x10 6 cells/mL in T cell media (RPMI-1640 (Gibco #11875093) containing 10% FBS (Bovogen #SFBS), sodium pyruvate (Gibco #11360070), nonessential amino acids (Gibco #11140050), HEPES (Gibco #15630130), Glutamax (Gibco #35050061), penicillin/streptomycin (Gibco #15140122)), stimulated with recombinant human IL-2 (100 U/mL; NIH) and SIINFEKL (10 ng/mL; Auspep) and plated onto the virus-coated plates at ~8x10 6 cells/well, before being incubated for 72 h at 37 o C. Expanded enAsCas12a KI/+ ;dCas9a-SAM KI/+ ;OT-I T/+ T cells were then washed and maintained in T cell media supplemented with 100 U/mL IL-2 at 0.5-1x10 6 cells/mL.All cell sorting was performed using an Aria II (BD Biosciences).

Statistical analyses
Statistical analyses (excluding the analysis of the Menuetto/Scherzo screens described above) were performed using Prism (v10.1.1,GraphPad).All statistical analyses performed reflect comparisons between distinct samples, rather than repeated measures.Differences between two groups were determined by Student's t-test, having been confirmed to conform with normality assumptions prior.Data are presented as means ± standard error of the mean (SEM), and statistical significance between groups is assessed by P-values, denoted by asterisks (*=P<0.05,**=P<0.01,and ns = no significant difference).Eμ-Myc T/+ ;enAsCas12a KI/+ ;Trp53 +/-cell lines (#205 #207) derived from the splenic tissue of the reconstituted mice.Control cell line #19 was derived from a double-transgenic Eμ-Myc T/+ ;enAsCas12a KI/+ lymphoma-burdened mouse.TRP53 stabilisation was induced via 24 h treatment with nutlin-3a (in the presence of QVD-O-Ph).β-ACTIN expression was used as a loading control.Figure 3. Application of Cas12a ultra-compact, genome-wide, multiplexed murinespecific pre-crRNA libraries.(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.Screens of 6 replicate transductions were performed for each library.(B,C) 4-way plots comparing the different arms of the screen samples for the Menuetto (B) and Scherzo (C) libraries.The y-axes compare S63845-treated samples with the input samples, and the x-axes compare 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 are indicated in light grey.Complete screen analyses can be found in Supplementary File 2.

Figure 4 .
Figure 4. Multiplexing of enAsCas12a and dCas9a-SAM in OT-I T cells.(A)Quantification of FACS analysis of enAsCas12a KI/+ ;dCas9a-SAM KI/+ ;OT-I T/+ T cells transduced with a Cd19-targeting sgRNA (BFP-tagged) for dCas9a-SAM-mediated gene expression and a Trp53-targeting pre-crRNA (GFP-tagged) for Cas12a-mediated gene-editing.~6% of cells from the sample transduced with both constructs were both GFP-and BFP-positive.Results are the averaged data from n=3 transductions.Error bars indicate SD. (B) Quantification of FACS analysis of the T cells transduced with lentiviral vectors expressing the Trp53-targeting crRNA and either a NTC sgRNA or the Cd19-targeting sgRNA.The Cd19-targeting sgRNA was able to induce strong CD19 expression in the double-transduced cells, as determined by intracellular antibody staining.Results represent data from n=3 T cell transductions.In this graph, mean is plotted and error bars represent the SEM.(C) NGS data from the GFP-positive andnegative populations (both CD19-positive) of T cells transduced with both the Trp53targeting pre-crRNA and the Cd19-targeting sgRNA.~50% of reads indicated a frameshift mutation had been generated in Trp53.Sequencing was performed for n=1 transduced T cell line.(D) Representative histogram (normalised to the mode) demonstrating CD19 upregulation in the multiplexed, sgCd19-transduced enAsCas12a KI/+ ;dCas9a KI/+ MDFs.CD19 expression for the empty vector transduced MDFs are shown in grey, crTrp53 in pink, and crBax/Bak in blue.(E) Western blots demonstrating TRP53, BAX, and BAK loss in the multiplexed enAsCas12a KI/+ ;dCas9a KI/+ MDFs (n=3).(F) Quantification of the western blots shown in G, demonstrating that while TRP53 and BAX loss were very efficient, BAK loss was incomplete in all samples.In this graph, the means are plotted and the error bars represent SD.