Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation

Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation View ORCID Profile Bo Gu , James M. Linton , Brice Graham Hendrickson , Hengyu Li , Ron Hadas , Gal Manella , Jan Gregrowicz , View ORCID Profile Michael B. Elowitz doi: https://doi.org/10.1101/2025.06.15.659618 Bo Gu 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bo Gu James M. Linton 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brice Graham Hendrickson 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hengyu Li 3 Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ron Hadas 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gal Manella 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan Gregrowicz 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael B. Elowitz 1 Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, CA 91125, USA 2 Howard Hughes Medical Institute, California Institute of Technology , Pasadena, CA 91125, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael B. Elowitz For correspondence: melowitz{at}caltech.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The ability to perturb multiple proteins simultaneously within the same cell is essential for understanding and re-engineering biological pathways. CRISPR-Cas12a mutants with inactivated DNAse but intact RNAse activity (dCas12a) retain the ability to process large CRISPR RNAs (crRNAs) arrays, enabling them to target multiple genomic loci in parallel. When coupled with transcriptional effector domains, these properties make Cas12a a promising platform for multi-locus transcriptional perturbation. However, current Cas12a-based CRISPRi systems exhibit limitations in processing of multi-crRNA arrays and transcriptional regulation. Here, we combine molecular and circuit-level engineering to develop a programmable Cas12a- based CRIPSRi system capable of strong, tunable, and simultaneous knockdown of six or more genes in a single cell without genomic DNA cleavage. We demonstrate the utility of this system by systematically perturbing a partially redundant set of Bone Morphogenetic Protein (BMP) receptors, enabling quantitative analysis of BMP signaling across diverse receptor configurations. Introduction Biological pathways often rely on families of partially redundant, interacting proteins. Gaining a mechanistic understanding of these pathways would be greatly facilitated by the ability to simultaneously perturb the expression of multiple proteins within the same cell in a controlled and predictable manner. While existing multiplexed genetic perturbation methods can readily target different genes across different cells, they generally fall short of enabling coordinated knockdown of multiple genes within the same cell. For example, reverse genetic studies predominantly rely on genetically modified cell lines or transgenic animals as model systems. Generation of these model systems is time-consuming and labor-intensive, involving the sequential introduction of multiple genetic modifications in order to avoid potential genotoxic effects or diminishing modification efficiency 1 , 2 . In forward genetic screens, large-scale combinatorial perturbations have been mostly restricted to yeast, aided by extensive strain collections with diverse genetic backgrounds 3 – 7 . In contrast, mammalian genetic screens using siRNA, shRNA, or other tools have largely been limited to single or, at most, pairwise perturbations 8 – 11 . More recently, with the advances in pooled genetic screens coupled to single- cell readout 12 , 13 , multiplexed genetic perturbations can occasionally be achieved but are still limited to up to three genetic perturbations per cell 14 , 15 . Thus, despite revolutionary advances in the last few decades, we lack convenient methods for simultaneous modulation of multiple proteins in single cells 16 – 21 . The CRISPR-Cas12a protein provides a promising platform for multiplexed genetic perturbation for three reasons. First, it possesses inherent RNase activity, allowing it to process a poly- crRNA array into individual crRNAs without accessory proteins 22 , 23 . Second, the direct repeat sequence of Cas12a is 19-nt, much shorter than its Cas9 counterpart, allowing the compact design and convenient synthesis of poly-crRNA arrays encoding a large number of crRNAs 22 . Third, DNAse-dead Cas12a (dCas12a) can be used as an RNA-guided DNA binder and further functionalized by fusing with diverse epigenetic effector domains 24 – 27 . Together, these features enable a compact, modular system for multiplexed genetic perturbation composed of a poly- crRNA array and dCas12a-effector fusion proteins ( Fig. 1A ). Despite these advantages, previous attempts to implement dCas12a-based multiplexed genetic perturbations have achieved limited success, largely due to several technical challenges, including (1) dilution or interference effects from dCas12a itself 28 ; (2) inefficient processing of poly-crRNA arrays 29 ; (3) reduced perturbation efficiency at constrained crRNA dosages ; 30 –; 32 ; and (4) suboptimal activity of effector domains 33 . Download figure Open in new tab Fig. 1. Development of Pathway Sculptor . (A) Schematic showing principles of dCas12a- based CRISPRi system. (B) Design of dCas12a-effector variants library composed of combinations of promoter variants, dCas12a variants, transcriptional effectors, and construct arrangement variants. (C) Experimental workflow for systematic dCas12a-effector library screening and multi-dimensional optimization. (D) Benchmark of Pathway Sculptor against existing dCas12a-based CRISPRi platform. Each data point denotes the median normalized GFP intensity of an independent experiment. Error bars denote the standard error of the means across three replicates. (E) RNA-seq read-counts comparing conditions transfected with non- targeting (NT) crNRA vs EF1a-targeting crRNA. n = 3 biologically independent RNA-seq experiments. Dots represent transcripts; star denotes Citrine transcript. In this study, we engineered a dCas12a-based CRISPRi system via systematic optimization of construct designs, effector domains, promoter selection, and component stability. The resulting system, dubbed, Pathway Sculptor, enables strong, specific, and durable multiplexed knockdown of at least N = 6 endogenous genes. Pathway Sculptor is robust across diverse cell types, plasmid configurations, and delivery approaches. We also developed methods for rapid engineering of stable cell lines expressing Pathway Sculptor in a titratable and reproducible manner. Using these cell lines, we achieved synchronous, tunable, and durable perturbation of endogenous genes, enabling studies with quantitative objectives. Finally, we applied Pathway Sculptor to reconfigure the Bone Morphogenetic Protein (BMP) receptor profile and uncovered a broad receptor expression regime in which whole-pathway activity remains sensitive. Results Development of Pathway Sculptor dCas12a-based CRISPRi systems have been shown to function effectively in common lab cell lines, but are typically analyzed at high, and potentially toxic, expression levels. This presents a challenge, as basic research or therapeutically-relevant CRISPR-based applications typically require limiting the expression of CRISPR components. For example, pooled CRISPR screening requires that sgRNAs encoding genetic perturbations be delivered at low viral titer to ensure library diversity 34 – 36 . In vivo studies typically use transgenic animal models with genomically integrated transgenes at defined loci to ensure genetic stability and avoid disrupting endogenous gene expression—conditions that constrain the intracellular levels of both dCas12a proteins and crRNAs. Similarly, therapeutic applications in primary cells are often hampered by inefficient delivery of CRISPR components, further restricting their intracellular concentration 31 , 37 , 38 . These limitations are exacerbated in settings that require multiplexed perturbations, where existing dCas12a-CRISPRi systems frequently underperform 28 , 31 , 32 , 39 . Together, these challenges provoke the question of whether the Cas12a system can be improved to allow operation even at low expression level. To address this question, we generated a library of 50 rationally-designed dCas12a-effector variants. Each library member incorporated one of three Cas12a variants, three plasmid configurations, three effector domains, four nuclear localization sequences, and used one of two possible domain arrangements ( Fig. 1B ). To systematically characterize the library, we engineered a reporter cell line carrying a genomically integrated destabilized Citrine fluorescent protein constitutively expressed from a EF1α promoter. We utilized a compact all-in-one construct design, allowing co-delivery of crRNAs targeting EF1α and one of the Cas12a-effector variants ( Fig. 1C ). Transient delivery of individual library members into the reporter cell line induced epigenetic silencing of the EF1α promoter, rapidly reducing fluorescence from the destabilized Citrine reporter, as measured by flow cytometry ( Fig. 1C ). Based on this screen, we developed design rules for an effective dCas12a-based CRISPRi system. First, the Cas12a variant hyperdCas12a, an LbCas12a mutant with enhanced DNA binding 30 , consistently outperformed existing AsdCas12a variants in mediating transcriptional repression ( Fig. S1A ). Second, crRNA abundance is a major limiting factor for knockdown efficiency ( Fig. S1B ). Third, fusing the ZIM3 KRAB effector to the N-terminus of dCas12a results in stronger repression than C-terminal fusions ( Fig. S1C ). These insights guided the development of an optimized Cas12a variant, comprising a CAG-driven hyperdCas12a with an N-terminal fusion of ZIM3 KRAB ( Fig. 3D ). This variant surpassed existing dCas12a-based CRISPRi platforms in the speed, magnitude, and durability of repression of a Citrine reporter ( Fig. 1D ). Whole-transcriptome RNA sequencing of cells transfected with EF1α promoter targeting crRNA showed a 10-fold decrease in Citrine expression across biological replicates compared to a negative control transfected with non-targeting crRNA ( Fig. 1E ). The downregulation of Citrine gene exceeds the differential expression of nearly all non-targeted genes. Taken together, our screen identified a new dCas12a-effector variant and an optimal expression construct with enhanced potency, persistence, and specificity over existing systems, serving as the basis for an improved dCas12a-based CRISPRi system for various applications. Because of its potential ability (demonstrated below) to simultaneously knockdown multiple genes within the same pathway, we termed this system Pathway Sculptor . Pathway Sculptor achieves potent, specific, durable multiplexed perturbation of endogenous genes A key enabling feature of dCas12a is its potential for multiplexed perturbation by a single construct. To assess this capability, we designed an all-in-one construct carrying poly-crRNAs as well as the optimized Cas12a effector, and tested whether it could simultaneously repress multiple endogenous genes. To test multiplexed knockdown, we selected a panel of CD surface proteins whose protein-level expression can be conveniently measured using cell surface staining followed by flow cytometry ( Fig. 2A ). These markers span a broad range of basal expression levels in HEK293T cells, allowing us to assess how knockdown efficacy depends on target abundance ( Fig. S2A ). Download figure Open in new tab Fig. 2. Pathway Sculptor achieves potent, specific, and prolonged multiplexed perturbation of endogenous genes. (A) Experimental workflow for multiplexed assessment of protein-level perturbation using Pathway Sculptor . WT HEK293T were transiently transfected with the all-in-one construct co-expressing poly-crRNA array targeting 4 selected CD surface proteins as well as sculptor protein. Cells were subject to surface antibody co-staining at varying time points after the initial transfection. (B) Flow cytometric measurement of CD markers surface level 144 hrs after transfection. x-axis label indicates perturbation conditions. Dot denotes median staining intensity normalized to conditions transfected with NT crNRA from an independent biological replicate. Bar heights and error bars denote the mean and s.e.m. across three independent biological replicates. (C) Differential expression analysis of three independent RNA-seqs comparing samples perturbed with CDarray vs NT crRNA. Dots denote individual genes. Genes with padj 1.73 are highlighted with red circles. (D) Flow cytometric measurement of CD protein level at varying time after initial transfection. Dot denotes median staining intensity normalized to conditions transfected with NT crNRA from an independent biological replicate. Bar heights and error bars denote the mean and s.e.m. across three independent biological replicates. Download figure Open in new tab Fig. 3. Pathway Sculptor is robust across diverse cell types and delivery approaches. (A) Flow cytometric measurement of synthetic reporter gene expression from a population of cells transfected with NT crRNA or crRNA targeting the promoter of each synthetic target. (B) Flow cytometric measurement of the expression levels of surface proteins, as indicated in Fig. 2A . All values are extracted from mcherry-gated population to selectively examine cells positively delivered. Dot denotes median staining intensity normalized to WT from independent biological replicates. Bar heights and error bars denote the mean and s.e.m. across n = 3 independent biological replicates. (C) Flow cytometric measurement of synthetic reporter gene expression. Transient delivery of the all-in-one knockdown constructs—encoding either individual crRNAs or poly-crRNA arrays targeting these CD markers—resulted in robust knockdown of all four targets (>70% reduction; Fig. 2B ) . Notably, the effect of repression of a given crRNA is not compromised in the context of poly-crRNA array ( Fig. 2B ), a phenomenon observed with previous Cas12a-CRISPRi systems 28 . To assess specificity, we performed RNA-seq on cells transfected with the poly-crRNA construct. Strikingly, the four targeted CD markers were the only genes significantly downregulated across the transcriptome ( Fig. 2C ). To evaluate the persistence of multiplexed gene repression, we performed longitudinal flow cytometry to track the expression of four CD proteins following the initial transient delivery of the all-in-one construct. Repression was sustained for at least ten days post-transfection ( Fig. 2D ). Taken together, these results showed that transient delivery of the Pathway Sculptor system could achieve potent, specific, and durable multiplexed repression of endogenous gene targets. Pathway Sculptor functions in diverse cell types with multiple delivery approaches An ideal CRISPRi platform should operate across a broad range of cell types. To test this, we first assessed repression of synthetic targets in the widely used U2OS and NIH3T3 cell lines. When delivered transiently via chemical transfection, the all-in-one construct ( Fig. 1C ) achieved strong repression of synthetic reporters in both cell types ( Fig. 3A , top and middle). We next turned to mouse embryonic stem cells (mESCs), which are valuable for basic research and applications, but more difficult to chemically transfect due to dense colony growth and sensitivity to chemical reagents. We delivered all-in-one constructs using electroporation. The overall transfected mCherry signal was low, likely reflecting inefficient delivery due to the large construct size. Nevertheless, we observed substantial repression of Citrine fluorescence within the mCherry-positive population ( Fig. 3A , bottom). Together, these results indicate that Pathway Sculptor functions effectively in diverse cell contexts. We also analyzed multiplexed gene repression in other cell types. using the same panel of human CD surface proteins above ( Fig. 2A ), we measured simultaneous repression of these targets in U2OS and K562 cells. Transient delivery of the all-in-one construct resulted in strong knockdown of all targeted CD proteins within the mCherry-positive population ( Fig. 3B , top and middle). To assess repression in a murine context, we designed a poly-crRNA array targeting three mouse CD proteins expressed at varying levels in mESCs ( Fig. S3A ). Electroporation of the all-in-one construct carrying this array led to robust repression of all three targets in the mCherry-positive population ( Fig. 3B , bottom). As expected, a non-targeting crRNA control produced minimal repression of all tested CD markers ( Fig. 3B ). Messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs) has matured into a clinically proven delivery modality 40 – 42 . To see whether the system could also function when delivered in LNPs, we encoded the Sculptor protein as mRNA and co-delivered it with chemically synthesized crRNAs targeting EF1α into a 293T Citrine-reporter cell line using LNPs. For comparison, we also delivered the all-in-one DNA construct via chemical transfection. We observed faster repression of Citrine fluorescence with mRNA delivery ( Fig. 3C , top), likely due to the higher transfection efficiency and more rapid protein production from transfected mRNA compared to DNA transfection. Over extended time courses, however, DNA-based delivery achieved stronger repression ( Fig. 3C , bottom), consistent with the transient, pulse-like nature of mRNA-based protein expression 43 . Taken together, these results demonstrate that Pathway Sculptor functions across multiple cell types and delivery approaches. Engineered Pathway Sculptor cell lines enable synchronous and tunable multiplexed genetic perturbation Engineered cell lines are widely adopted in CRISPR-based perturbation tools and have enabled many important discoveries 13 , 44 – 47 . Stable expression of these systems at lower levels reduces cellular stress and toxicity, allows uniform silencing across cells, and permits reproducible, tunable control of perturbation strengths 46 , 48 . A stable cell line expressing Pathway Sculptor should provide similar advantages, and thereby enable engineering of transgenic animals and facilitate high-throughput functional genomics screens. However, engineering of stable cell lines has been historically time-consuming and labor-intensive. Further, genomically-integrated transgenes are often subject to epigenetic silencing. To address these challenges, we devised two strategies: (1) random integration using the iON piggyBac transposon 49 ( Fig. 4A ), and (2) site-specific integration in a safe-harbor locus 50 ( Fig. 4B ). Both approaches incorporate doxycycline-inducible expression of Sculptor and in different ways couple genomic integration with transgene expression, accelerating antibiotic selection and allowing rapid derivation of stable cell lines. Additionally, both designs feature bidirectional promoter architectures to minimize transcriptional crosstalk ( Fig. 4A and B ), a common source of leaky expression in other Dox-inducible systems 51 . Use of the genomic safe-harbor locus 50 in the second strategy also mitigates transgene silencing, supporting long-term and reproducible expression of the Sculptor system. Download figure Open in new tab Fig. 4. Rapidly engineered Pathway sculptor cells can achieve synchronous and tunable multiplexed genetic perturbation. (A–B) Schematics of two strategies for Pathway Sculptor cell line engineering: PiggyBac-based and Safe-harbor locus site directed targeting of Dox- inducible Pathway Sculptor construct. (C) Experimental workflow for the comparison of an all-in- one system with an engineered sculptor cell line. Stable Pathway Sculptor cell line were derived by co-transfection of plasmids encoding piggybac transposase and iON-PB-sculptor construct carrying Dox-inducible sculptor protein and constitutive poly-crRNA CD targeting array, followed by antibiotic selection for 96 hrs. Engineered cell line and WT cell transiently transfected with all-in-one sculptor construct were subject to multiplexed antibody co-staining followed by flow cytometric measurement. (D) Flow cytometric measurement of CD protein level at varying time points after Dox induction or after initial transfection of all-in-one construct, in the case of engineered stable cell line and WT cells, respectively. y-values represent kernel density estimates of the probability density function. (E) Schematics of two titration strategies deployed in engineered Pathway Sculptor cell lines. Top: titratability is achieved through administration of different concentrations of Dox, or 4-ED. Bottom: titratability is achieved through administration of saturated concentration of Dox for different duration of time. (F-G) Multiplexed flow cytometric measurement revealed tunable CD protein level with different titration strategies. (H) Schematics of the designs of different Pathway Sculptor constructs with stability-enhancement features. (I) Multiplexed flow cytometric measurement of mouse CD protein knockdown in engineered mESC cell lines carrying different stability-enhanced versions of Pathway Sculptor upon 48 hrs of Dox induction. In all plots, dot denotes median staining intensity normalized to WT from independent biological replicates. Bar heights and error bars denote the mean and s.e.m. across three independent biological replicates. Heterogeneity in genetic perturbation across a cell population poses a major challenge for interpreting the outcomes of perturbation experiments. To evaluate whether the engineered Sculptor cell line could mitigate this heterogeneity, we generated HEK293 cell lines stably expressing the Pathway Sculptor system using the iON-PB strategy ( Fig. 4C , top). Specifically, we compared the kinetics and uniformity of CD protein repression over 120 hours ( Fig. 4C , top) following Dox induction against a transient delivery experiment using an all-in-one construct ( Fig. 4C , bottom). In both settings, we observed progressive loss of all three targeted CD proteins ( Fig. 4D ). Notably, at each time point, the engineered stable cell line exhibited a more homogeneous distribution of marker expression compared to the transiently transfected cells ( Fig. 4D ), indicating reduced perturbation heterogeneity. These results demonstrate that engineered Pathway Sculptor cell lines enable uniform multi-gene repression across the population. The engineered Pathway Sculptor cell lines also enabled tunable control of expression through three orthogonal strategies: (1) modulating Sculptor protein and/or crRNA levels (concentration tuning), (2) varying the duration of repression (duration tuning), and (3) incorporating mismatches into crRNAs to modulate binding strength (affinity tuning). To achieve concentration tuning, we treated polyclonal HEK293T cells with increasing concentrations of Dox or its analog, 4-epidoxycycline (4-ED). While Dox induced all-or-none expression of sculptor, 4-ED allowed robust titration of sculptor across two orders of magnitude ( Fig. S4A ). To different extents, this titration achieved graded knockdown of the four targeted CD markers ( Fig. 4F ). CD55, CD184, and CD201 exhibited a tunability range of up to 5-fold, whereas CD321 showed a more modest 1.5-fold reduction, likely due in part to its high basal expression. Tuning the duration of Dox exposure similarly led to graded repression across all markers ( Fig. 4G ), with CD321 again showing a more limited response. For affinity tuning, we introduced single mismatches at different positions of an EF1α-targeting crRNA to modulate its binding strength. This approach enabled fine-tuned repression of a Citrine reporter upon transfection into engineered cells carrying an additional genomically integrated EF1α-Citrine transgene ( Fig. S4D ). Together, these results demonstrate that engineered Pathway Sculptor cell lines support tunable multi- gene regulation with flexible and orthogonal control mechanisms. mESCs exhibit a short doubling time of ∼12 hours, leading to relatively rapid dilution of intracellular molecules and reduced steady-state concentrations. This presents a challenge for CRISPR-based epigenetic modulation systems, whose efficacy depends on maintaining sufficient intracellular levels of effector proteins and guide RNAs. Partly as a result, there have been relatively few studies using CRISPRi in mESCs. To test our cell line engineering strategy in mESC, we generated stable Pathway Sculptor mESCs using the iON-PB strategy with a constitutively expressed poly-crRNA array targeting mCDs. Unlike the case in engineered HEK293T cells, titrating Dox or 4-ED failed to produce tunable Sculptor expression in mESCs ( Fig. S4B ). We therefore explored the duration-titration scheme successfully deployed in HEK293T cell lines. As expected, all three mCD targets were repressed in a tunable manner with varying durations of Dox exposure ( Fig. S4C ). However, the overall repression in mESCs was much weaker than in HEK293T cells, likely due to low intracellular abundance of the Pathway Sculptor components. To address these issues, we enhanced the stability of the crRNA and the engineered dCas12a effector. crRNAs are prone to degradation by endogenous exonucleases, which limits their steady-state intracellular concentration 52 – 54 . Circularizing RNA can mitigate this degradation by protecting against exonuclease activity 55 , 56 . We therefore flanked the crRNA or poly-crRNA array with the Tornado system—a ribozyme-based, self-circularizing RNA platform—to promote crRNA stabilization 57 . In parallel, we increased dCas12a protein stability by incorporating the XTEN linker, a peptide known to prolong protein half-life 58 . This approach has been successfully used in Cas9-based CRISPRi systems and recently adapted for Cas12a 28 , 59 . We evaluated the effects of these enhancements by incorporating these features into engineered mESCs ( Fig. 4H ). As expected, the unmodified construct ( Sculptor _v1) showed modest repression ( Fig. 4I ). In contrast, both stability-enhanced versions ( Sculptor _v2 and v3) produced markedly stronger repression, and their combination ( Sculptor _v4) yielded a synergistic effect ( Fig. 4I ). Notably, the repression of CD31 is not as strong as the other CD proteins, likely due to its high basal expression level. Taken together, these results showed that engineered Pathway Sculptor cell lines allow synchronous, tunable, and simultaneous knockdown of multiple endogenous genes. Pathway Sculptor enables quantitative analyses of pathway-level BMP signaling responses With its enhanced potency and multiplexing capability, Pathway Sculptor enables perturbation at the level of multi-gene modules—rather than individual genes—opening new possibilities for systematically mapping gene modules–phenotype relationships. The Bone Morphogenetic Protein (BMP) pathway is a crucial signaling pathway involved in various biological processes, including embryonic development, tissue homeostasis, and regeneration 60 . The BMP pathway comprises multiple ligand and receptor variants that interact promiscuously with one another to achieve their functions. Mammalian species have over 20 distinct ligands, 4 type I receptor subunits (BMPR1A, BMPR1B, ACVR1, and ALK1), and 3 type II receptor subunits (BMPR2, ACVR2A, and ACVR2B). These components can combine in various ways to form hundreds of receptor-ligand signaling complexes, each consisting of 2 type I and 2 type II receptors bound to a dimeric ligand 61 , 62 . Complete functional abrogation of BMP pathway activity would enable systematic investigation of its role, both alone and in combination with other signaling pathways. However, due to the combinatorial nature of ligand–receptor interactions, traditional approaches that perturb individual ligands or receptors often leave residual pathway activity. Achieving full pathway suppression therefore requires highly multiplexed receptor perturbation—a task well suited for Pathway Sculptor . We selected HEK293T cells expressing six of seven BMP receptors according to publicly available dataset. Complete knockdown of all six of these BMP receptors is challenging for canonical genetic perturbation methods. We therefore designed and generated a CAG promoter-driven poly-crRNA array (hereafter termed BMPR mega-array) harboring 32 crRNAs targeting all seven BMP receptors (each targeted by 2-5 crRNAs). We transiently delivered a construct with CAG promoter-driven BMPR mega-array alongside a construct expressing the dCas12a-effector protein into an engineered HEK293T cell line carrying a BMP signaling reporter responsive to a broad range of BMP ligands 63 ( Fig. 5A ). We assessed BMP receptor knockdown at the mRNA level using RNA-seq and evaluated functional signaling responses using a ligand-stimulation assay ( Fig. 5A ). RNA-seq and flow cytometry respectively revealed strong knockdown of all seven BMP receptors ( Fig. S5A ) and reduced signaling responses to all five tested BMP ligands ( Fig. 5B ). Download figure Open in new tab Fig. 5. Pathway Sculptor enables the cellular reconstitution of novel BMP signaling responses. (A) Experimental workflow for reconfiguration of BMP receptor profiles with transiently delivered Pathway Sculptor . HEK239T cell line carrying a BMP signaling reporter are co-transfected with plasmid expressing sculptor protein and a poly-crRNA array harboring 32 crRNAs targeting all seven human BMP receptors. Functional and mRNA-level assessment were performed 96 hrs after transfection. Pair of type I (Acvr1) and type II (Acvr2b) mouse BMP receptors were subsequently introduced to the cells, followed by functional assessment of the reconstitution of BMP signaling response. (B) Flow cytometric measurement of Ctrine reporter expression in response to five canonical BMP ligands, normalized to unstimulated controls, in wild-type and Pathway Sculptor –perturbed cells. (C) Reconstituted BMP signaling response depends on the delivered mRNA dosage encoding mouse BMP receptors. (D) Experimental workflow for BMP receptor reconfiguration using engineered Pathway Sculptor cells. HEK293T cells carrying a BMP-responsive reporter were modified to express a single-copy, Dox-inducible Sculptor protein and a constitutive poly-crRNA array targeting all seven human BMP receptors (32 guides total). Functional assays were performed under varying conditions of endogenous receptor titration. (E-F) Endogenous BMP signaling response sensitively depends on the endogenous dosages of BMP receptors. In all plots, dot denotes median staining intensity normalized to WT from independent biological replicates. Bar heights and error bars denote the mean and s.e.m. across three independent biological replicates. The combinatorial expression profile of the BMP receptor determines the cell’s ability to perceive ligands 64 . In principle, HEK293T cells depleted of BMP receptors provide a clean “blank-slate” genetic background for reconstitution of specific receptor configurations. To explore this, we designed an add-back experiment in which two specific BMP receptors, Acvr1 and Acvr2b, were ectopically expressed in the receptor-depleted cell line, followed by stimulation with their likely cognate ligand, BMP-9 ( Fig. 5A ). As expected, this resulted in functional restoration of the signaling response ( Fig. 5C ). Strikingly, the magnitude of the reconstituted response exhibited a dose-dependency to the level of ectopic receptor expression ( Fig. 5C ), suggesting that variation in receptor expression levels could be a determinant of signaling amplitude in natural contexts. We next sought to understand whether the dose-dependent effect of receptor expression level on signaling can occur in a physiologically relevant expression regime and whether it generalizes to other receptors. We employed the site-specific targeting approach ( Fig. 4B ) to stably integrate a single-copy, Dox-inducible Pathway Sculptor module as well as a constitutively expressed BMPR mega-array ( Fig. 5D ). By modulating Dox concentration or exposure time, we achieved graded pathway-level responses to BMP ligand stimulation ( Fig. 5E and F ). These results suggest that Pathway Sculptor , even when expressed at limiting molecular concentrations, can quantitatively modulate the dosage of endogenous BMP receptors and elicit a corresponding dosage-dependent signaling response. More broadly, this system could be used to reconstitute physiologically relevant receptor configurations in diverse cell contexts. Discussion Non-cleaving genetic perturbation platforms minimize the risk of genotoxicity and reduce off- target effects, while potentially enabling reversible or multiplexed control of gene expression 44 , 59 , 65 – 70 . These platforms vary in their compactness as well as in potency, multiplexability, and durability of perturbations. Previous tools have been optimized for some of these features at the expense of others. For example, siRNA and shRNA are potent and compact, but offer limited multiplexing capabilities and require continuous reagent delivery to maintain prolonged knockdown. Similarly, CRISPR-Cas9-based CRISPRi systems excel in potency and compactness and, when repurposed, can induce irreversible gene silencing. Achieving multiplexed genetic perturbation with these systems, however, requires additional components. Pathway Sculptor exceeds both systems in all three features, making it a useful candidate for biotechnological and therapeutic applications. Its key differentiating features include the ZIM3 KRAB effector domain, the choice of nuclear localization sequence, inclusion of stabilizing feature, optimization of the crRNA expression unit, the design of the constructs and, in some cases, the choice of delivery or cell engineering strategies. Through integrative refinement of these elements, we obtained synergistic improvements in performance. The best version of the system now outperforms comparable tools across a wide range of cellular contexts. The system has limitations. First, we observed gene-to-gene variability in knockdown efficiency, with certain genes exhibiting substantially stronger repression than others. This could arise from variability in crRNA quality, which can affect the duration and abundance of recruited repressive machinery at the target locus. Alternatively, it may reflect differences in local chromatin context. Second, knockdown efficiency also varies between cell contexts. Sculptor relies on endogenous silencing machinery to mediate gene repression. As a result, the abundance or activity of these factors can influence knock-down efficiency. For instance, the ZIM3-KRAB domain recruits repression complexes by interacting with TRIM28 71 ; thus, cells with higher TRIM28 expression may exhibit stronger knockdown than those with lower TRIM28 levels. The ability to strongly and durably knock down multiple genes within a family will open up the ability to analyze different configurations of complex protein families. For example, in signaling pathways, including BMP, it is of interest to understand how different combinations of receptors process ligand inputs. Pathway Sculptor should open up the possibility of systematically analyzing different configurations of receptors or other components of protein families. Methods Tissue culture and cell lines HEK293T cells were cultured in DMEM (Thermo Fisher Scientific) + 10% FBS (Avantor® Seradigm) + Pen Strep L-glutamine and passaged every 2-3 days. U2OS cells were cultured in DMEM (Thermo Fisher Scientific) + 10% FBS (lot #) and passaged every 2-3 days. K562 Cells were grown in RPMI medium 1640 (ThermoFisher) + GlutaMAX (ThermoFisher) + 10% heat inactivated FBS (Invitrogen) + pen/strep (ThermoFisher) + L-glutamine (ThermoFisher) + sodium pyruvate (ThermoFisher). Cells were maintained at the density of around 1x10^6 cells/ml and passaged every 2-3 days. E14 mES cells (ATCC cat. No. CRL-1821) were cultured in medium containing DMEM (Sigma), 15% ES cell qualified FBS (Gibco), 1x MEM non-essential amino acids (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 100 μM B-mercaptoethanol (Thermo Fisher Scientific), 1x penicillin-streptomycin-L-glutamine (Thermo Fisher Scientific) and 1000 U/mL leukemia inhibitory factor (Millipore). Cells were maintained on polystyrene (Falcon) plates coated with 0.1% gelatin (Sigma) at 37 C and 5% CO2 and passaged every 48 hrs. NIH3T3 Reverba-Venus-NLS-PEST cells ( https://pubmed.ncbi.nlm.nih.gov/15550250/ ) were a gift from Prof. Gad Asher, and were cultured in DMEM (Thermo Fisher Scientific) + 10% FBS (Avantor® Seradigm) + Pen Strep L-glutamine and passaged every 4-5 days. All cells are routinely tested for mycoplasma contamination. Library preparation for RNA-seq mRNA were extracted from 96-well plate using Direct-zol-96 RNA Kits (Zymoresearch Cat# R2055). 50ng of extracted mRNA from each sample were used as inputs for downstream NGS library preparation. mRNA-seq library were prepared in 96-well format with a modified 3’Pool-seq protocol ( https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-020-6478-3#Sec9 ). In brief, reverse transcription reaction were prepared by mixing input RNA with 1 μl Indexed RT Primer (10 μM), 1 μl 10 mM dNTP Mix (New England Biolabs Cat# N0447S),1 μl diluted ERCC Spike-In Mix 1 (0.004 μL stock ERCC per μg RNA, ThermoFisher Cat# 4456740), 3.6 ul of 5x RT buffer (ThermoFisher Cat# EP0752), 0.5 ul of RNAse inhibitor (Thermofisher Cat# EO0381), 1 ul Maxima RT H minus (Thermofisher Cat# EP0752), 2.5 ul 10 uM Template Switching Oligo into a 18 ul reaction. Reverse transcription was carried out in a thermocycler with program described in 3’Pool-seq protocol. Samples from each row of 96-well plate were pooled (column pooling) by mixing an equal volume of each Reverse Transcription reaction into a new well at a total volume of 20 μl. Residual primers were then degraded with the addition of 1 μl Exonuclease I (New England Biolabs) and incubated at 37 °C for 45 min followed by denaturation at 92 °C for 15 min. Subsequent cDNA amplification, tagmentation, and row pooling was performed following 3’Pool- seq protocol. Finally, 20 ul of pooled NGS library were subject to Gel-based size selection using E-Gel EX Agarose Gel (ThermoFisher Cat# G401001) to enrich for fragments with size range between 200-1000 bp and eluted in 15 ul. Eluted pooled NGS library were examined in an Agilent TapeStation 4200 (Agilent Technologies) to determine average fragment sizes. Library concentration was quantified in a Qubit 3.0 Fluorometer (Life Technologies). NGS library molarity was then calculated using 660 g/mol per base-pair as a molecular weight. NGS library were diluted to 2 nM, denatured in 0.2N NaOH, and loaded onto Element AVITI sequencer following Element Biosciences Cloudbreak Sequencing user guide. Flow cytometry Cells for assessment were trypsinized with 40 μL of 0.05% trypsin-EDTA (Gibco) for 1 minute at room temperature, and subsequently resuspended in 100 μL of Hanks’ Balanced Salt Solution(HBSS) containing 2.5mg/ml bovine serum albumin (BSA), 1mM ethylenediaminetetraacetic acid (EDTA), and/or 4 units/ml DNase I (NEB). Cells were then filtered through a 40 μm cell strainer (Falcon™) or a 96-well plate cell strainer (Millipore) and analyzed by flow cytometry (CytoFLEX, Beckman Coulter or ZE5, Bio-Rad). RT-qPCR Total RNA was harvested from cell lysate using the RNeasy mini kit (QIAGEN) and cDNA was generated from 1μg of RNA using the iScript cDNA synthesis kit (BioRad) following the manufacturer’s instructions. Primers and probes for specific genes were purchased from IDT. Reactions were performed using 1:40 dilution of the cDNA synthesis product with either IQ SYBR Green Supermix or SsoAdvanced Universal probes Supermix (BioRad). Cycling was carried out on a BioRad CFX96 thermocycler using an initial denaturing incubation of 95° for 3 min followed by 39 cycles of (95°C for 15 s, followed by 60°C for 30 s). Each condition was assessed with three biological repeats and each reaction was run at least in triplicate. Lentivirus preparation To prepare lentivirus, HEK293T cells were plated on 2x10 cm dishes with 6,000,000 cells per dish. Cells were co-transfected the following day with 8.3 ug of lentiviral transfer plasmid, 5.7 ug of Gag/Pol, and 2.2 ug of pLP2, and 3.1 ug of VSVg packaging plasmids using lipofectamine 3000 following manufacture’s instructions ( assets.thermofisher.com/TFS- Assets/LSG/manuals/lipofectamine3000_protocol.pdf ). Media was harvested at 48 hours after transfection and stored at -80 C until further use. LNP encapsulation We adopted a previously described 4 component LNP formulation 72 (4A3-SC8, DOPE, Cholesterol, DMG-PEG) that enables transfection of cells in culture and in vivo . To prepare the lipid stock solutions, a full 25 mg tube of 4A3-SC8 compound was dissolved in 167 μL of pure ethanol to yield a 150 mg/mL stock solution. Separately, 10 mg of DOPE was dissolved in 1.0 mL of pure ethanol to produce a 10 mg/mL stock solution (alternatively, 100 mg in 10 mL ethanol). Similarly, 10 mg of cholesterol was dissolved in 1.0 mL of pure ethanol (or 50 mg in 5 mL ethanol), and 10 mg of DMG-PEG was dissolved in 1.0 mL of ethanol (or 50 mg in 5 mL ethanol), resulting in 10 mg/mL stock solutions for each. A 20 mM working lipid mixture was prepared by combining 6.7 μL of the 4A3-SC8 solution (23.8%, 4.76 mM), 50.7 μL of the DOPE/DSPC solution (23.8%, 4.76 mM), 52.7 μL of the cholesterol solution (47.6%, 9.52 mM), and 34.2 μL of the DMG-PEG solution (4.8%, 0.96 mM). The working lipid mixture was used to encapsulate plasmid DNAs in the lipid nanoparticle (LNP) formulation. Initially, the lipid mixture was equilibrated at room temperature for at least 5 minutes and then vortexed at speed 10 for 5 seconds. A lipid mastermix was subsequently prepared by mixing 12 µL of the lipid mixture with 18 µL of 200-proof ethanol, yielding 30 µL per reaction. This mastermix was distributed into individual tubes. Each RNA mixture was prepared by combining 40 µL of plasmid DNA solution (250 ng/µL; total of 10 µg) with 32 µL of nuclease-free water and 18 µL of 50 mM citrate buffer, yielding a total volume of 90 µL per reaction. To assemble the LNPs, 30 µL of the lipid mastermix was placed on a Vortex-Genie 2 vortex mixer set at speed level 1. While vortexing, 90 µL of the RNA mixture was rapidly pipetted into the lipid mastermix in a single action, and vortexing was continued for 30 seconds. The resulting dispersion was incubated at room temperature for 5 minutes, and dialysis was initiated within 15 minutes of mixing. Dialysis was performed using Pur-A-Lyzer Midi 3500 dialysis tubes. Each tube was preconditioned by adding 900 µL of water, incubating for 5 minutes, and then removing the water. Approximately 120 µL of each LNP sample was transferred into the dialysis tubes, which were placed into a holder and submerged in 1× PBS within a beaker. Dialysis was conducted either for 1 hour at room temperature or overnight at 4°C. After dialysis, each sample was transferred into an RNase-free 1.5 mL microcentrifuge tube, and the final volume was measured. Samples were adjusted to a total volume of 500 µL by adding the appropriate volume of 1× PBS. All samples were stored at 4°C. Quantifications and statistical analyses Flow cytometry data analyses: Flow cytometry data was analyzed in MATLAB using a custom software (EasyFlow: GitHub - AntebiLab/easyflow: Matlab Based Flow Cytometry Analysis Tool). Events collected from flow cytometry experiments were first gated based on forward vs. side scatter to select for cells, followed by gating based on forward scatter area vs forward height, to select for single cells. mRNA-seq analyses: Read de-multiplexing was performed with Bases2Fastq, a standard software package used by Element Bioscience system. Reads were aligned to a custom reference genome GRCh38.103 using STAR (2.7.8a) ( https://www.sciencedirect.com/science/article/pii/S009286742300689X?via%3Dihub#bib78 ) with the ENCODE standard options except “--outFilterScoreMinOverLread 0.3 -- outFilterMatchNminOverLread 0.3 --outFilterMismatchNmax 20 --outFilterMismatchNoverLmax 0.3 --alignSJoverhangMin 5 --alignSJDBoverhangMin 3”. Uniquely mapped reads that overlap with genes were counted using HTSeq-count (0.13.5) ( https://www.sciencedirect.com/science/article/pii/S009286742300689X?via%3Dihub#bib79 ) with default settings except “-m intersection-strict”. To normalize for differences in sequencing depth across samples, we rescaled gene counts to counts per million (CPM). Fold-change and adjusted p values were calculated using the R package DESeq2 ( https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8 ). Cell surface staining Cells for assessment were dissociated with accutase (Stemcell Technologies ?Cat# 07920) for 5min at RT and neutralized with 200 μL of Hanks’ Balanced Salt Solution(HBSS) containing 2.5mg/ml bovine serum albumin (BSA), 1mM ethylenediaminetetraacetic acid (EDTA), and/or 4 units/ml DNase I (NEB). Dissociated cells were filtered with a 40 um cell strainer and spun down at 300g for 5 min in V-bottom 96-well plate. Cells were then resuspended with flow buffer containing corresponding 1:500 diluted FP-conjugated antibodies targeting CD markers for 1 hr at RT on a belly dancer. After staining, cells were spun down and washed with flow buffer once before loading onto cytoflex machine for fluorescence measurements. Cell line engineering Piggybac-based random integration: piggybac transposons and hyperPBase/SuperPBase (system bioscienes Cat# PB210PA-1) were co-delivered into cells with either chemical transfection or encapsulated in LNP. For all cell types except mESC, SuperPBase were used as transposase, while hyperPBase were used for mESC due to its superior transposition efficiency. Cells were subject to antibiotic selection 48 hrs post-delivery and were continuously cultured in selection media for 3-7 days (cell bottlenecking), depending on the administered antibiotics. A positive control condition where either the transposon or transposase were omitted were used to indicate the completion of cell bottlenecking. Specifically for mESC, monoclones with appropriate expression level were derived by sorting single cells with flow cytometer and expanded for 1 week. For all other cell types, polyclonal population derived from antibiotic- based bottlenecking were directly used for experiments. Safe-harbor locus targeting: PDG458 (Addgene Plasmid #100900) with sgRNA targeting rosa/AAVS locus and donor plasmids with corresponding homology arms flanking sculptor and rtta were co-delivered into cells with either chemical transfection or LNP. Cells were subject to antibiotic selection 48 hrs post-delivery and were continuously cultured in selection media for 3- 7 days (cell bottlenecking), depending on the administered antibiotics. A positive control condition where either the Cas9 plasmid or donor plasmid were omitted in the delivery were used to indicate the completion of cell bottlenecking. Specifically for mESC, monoclones with appropriate expression level were derived by sorting single cells with flow cytometer and expanded for 1 week. For all other cell types, polyclonal population derived from antibiotic- based bottlenecking were directly used for experiments. We note that, as opposed to a PB- based random integration strategy, targeted strategy yields a relatively homogeneous population and therefore, for most of the cases, monoclonal derivation can be omitted. BMP ligand stimulation The BMP reporter cell line(s), both parental and Pathway Sculpter integrated, were plated at 40% confluency in 96 well plates and cultured under standard conditions (above) for 12 hr. Media was then replaced and ligand(s) were added at specified concentrations. Approximately, 24 hr after ligand addition, cells were prepared for flow cytometry by washing with PBS and lifting from the plate using trypsin and incubations of 5 min at 37°C. Protease activity was quenched by re-suspending the cells in HBSS with 2.5mg/ml Bovine Serum Albumin (BSA). Cells were then filtered with a 40μm mesh and analyzed by flow cytometry using a CytoFLEX S (Beckman Coulture). All recombinant BMP ligands were acquired from R&D Systems (bio- techne) and reconstituted as recommended by the manufacturer. Author contributions B.G. and M.B.E. conceived and designed the study. M.B.E. directed and supervised the study. B.G., J.M.L and M.B.E. directed and supervised experiments corresponding to Fig. 5 . J.G. performed mRNA-LNP experiments. G.M. and R.H. assisted with or offered guidance regarding experiments corresponding to Fig. 3 . B.G.H. assisted with molecular clonings and longitudinal flow cytometry experiments. H.L. assisted with experiments corresponding to supplementary figures. B.G. and J.M.L. analyzed data. B.G. and M.B.E. wrote the manuscript with input from all authors. Competing interests Patent applications related to this work have been filed by the California Institute of Technology. M.B.E. is a scientific advisory board member or consultant at TeraCyte, Plasmidsaurus, Asymptote Genetic Medicines, and Spatial Genomics. Data and code availability Data needed to evaluate the conclusions are available in the paper, the extended data figures, or the Supplementary Materials. Raw data and computer code used for data analysis are available from the corresponding author upon request. Materials and correspondence All material requests and correspondence should be addressed to M.B.E. Acknowledgement We thank Dongyang Li, Shiyu Xia, Yodai Takei, Lukas Moeller, Andrew Lu for advice on experimental design, technical support, insightful discussions, or critical proofreading of the manuscript; Inna-Marie Strazhnik for graphical design; Leah Santat, Jo Leonardo, and Rui Malinowski for administrative support. This research was supported by the National Institutes of Health (EB030015). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M.B.E. is a Howard Hughes Medical Institute Investigator. B.G. is supported by the Damon Runyon Fellowship (DRG2441-21). G. M. is supported by the Human Frontiers Science Program (HFSP.MANELLA). J.G. is supported by the Boehringer Ingelheim Fonds PhD fellowship. Funder Information Declared Damon Runyon Cancer Research Foundation, https://ror.org/01gd7b947 , DRG-#2441-21 International Human Frontier Science Program Organization , HFSP.MANELLA References 1. ↵ Kosicki , M. , Tomberg , K. & Bradley , A . Repair of double-strand breaks induced by CRISPR- Cas9 leads to large deletions and complex rearrangements . Nat. Biotechnol . 36 , 765 – 771 ( 2018 ). OpenUrl CrossRef PubMed 2. ↵ Cullot , G. et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations . Nat. Commun . 10 , 1136 ( 2019 ). OpenUrl CrossRef PubMed 3. ↵ Kuzmin , E. et al. Systematic analysis of complex genetic interactions . Science 360 , ( 2018 ). 4. Taylor , M. B. & Ehrenreich , I. M . Genetic interactions involving five or more genes contribute to a complex trait in yeast . PLoS Genet . 10 , e1004324 ( 2014 ). OpenUrl CrossRef PubMed 5. Taylor , M. B. & Ehrenreich , I. M . Transcriptional derepression uncovers cryptic higher-order genetic interactions . PLoS Genet . 11 , e1005606 ( 2015 ). OpenUrl CrossRef PubMed 6. Celaj , A. et al. Highly combinatorial genetic interaction analysis reveals a multi-drug transporter influence network . Cell Syst . 10 , 25 – 38 .e10 ( 2020 ). OpenUrl CrossRef PubMed 7. ↵ Costanzo , M. et al. A global genetic interaction network maps a wiring diagram of cellular function . Science 353 , aaf1420–aaf1420 ( 2016 ). 8. ↵ Silva , J. M. et al. Profiling essential genes in human mammary cells by multiplex RNAi screening . Science 319 , 617 – 620 ( 2008 ). OpenUrl Abstract / FREE Full Text 9. Bassik , M. C. et al. Rapid creation and quantitative monitoring of high coverage shRNA libraries . Nat. Methods 6 , 443 – 445 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 10. Carette , J. E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing . Nat. Biotechnol . 29 , 542 – 546 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 11. ↵ Bassik , M. C. et al. A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility . Cell 152 , 909 – 922 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 12. ↵ Dixit , A. et al. Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens . Cell 167 , 1853 – 1866 .e17 ( 2016 ). OpenUrl CrossRef PubMed 13. ↵ Adamson , B. et al. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response . Cell 167 , 1867 – 1882 .e21 ( 2016 ). OpenUrl CrossRef PubMed 14. ↵ Wong , A. S. L. , Choi , G. C. G. , Cheng , A. A. , Purcell , O. & Lu , T. K . Massively parallel high- order combinatorial genetics in human cells . Nat. Biotechnol . 33 , 952 – 961 ( 2015 ). OpenUrl CrossRef PubMed 15. ↵ Zhou , P. et al. A Three-Way combinatorial CRISPR screen for analyzing interactions among druggable targets . Cell Rep . 32 , 108020 ( 2020 ). 16. ↵ Grünenfelder , B. & Winzeler , E. A . Treasures and traps in genome-wide data sets: case examples from yeast . Nat. Rev. Genet . 3 , 653 – 661 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 17. Adams , M. D. & Sekelsky , J. J . From sequence to phenotype: reverse genetics in Drosophila melanogaster . Nat. Rev. Genet . 3 , 189 – 198 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 18. Carpenter , A. E. & Sabatini , D. M . Systematic genome-wide screens of gene function . Nat. Rev. Genet . 5 , 11 – 22 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 19. Boutros , M. & Ahringer , J . The art and design of genetic screens: RNA interference . Nat. Rev. Genet . 9 , 554 – 566 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 20. Friedman , A. & Perrimon , N . Genome-wide high-throughput screens in functional genomics . Curr. Opin. Genet. Dev . 14 , 470 – 476 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 21. ↵ Mohr , S. , Bakal , C. & Perrimon , N . Genomic screening with RNAi: results and challenges . Annu. Rev. Biochem . 79 , 37 – 64 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ Fonfara , I. , Richter , H. , Bratovič , M. , Le Rhun , A. & Charpentier , E . The CRISPR- associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA . Nature 532 , 517 – 521 ( 2016 ). OpenUrl CrossRef PubMed 23. ↵ Zetsche , B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . Cell 163 , 759 – 771 ( 2015 ). OpenUrl CrossRef PubMed 24. ↵ Tak , Y. G. , et al. Inducible, tunable and multiplex human gene regulation using CRISPR- Cpf1-based transcription factors . bioRxiv ( 2017 ) doi: 10.1101/150656 . OpenUrl Abstract / FREE Full Text 25. Tang , X. et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants . Nat. Plants 3 , 17103 ( 2017 ). 26. Zhang , X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems . Protein Cell ( 2017 ) doi: 10.1007/s13238-017-0491-6 . OpenUrl CrossRef 27. ↵ Kleinstiver , B. P. et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing . Nat. Biotechnol . 37 , 276 – 282 ( 2019 ). OpenUrl CrossRef PubMed 28. ↵ Hsiung , C. C.-S. et al. Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations . Nat. Biotechnol . ( 2024 ) doi: 10.1038/s41587-024-02224-0 . OpenUrl CrossRef 29. ↵ Yang , L.-Z. et al. CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells . Nat Methods 21 , 1646 – 1657 ( 2024 ). OpenUrl CrossRef PubMed 30. ↵ Guo , L. Y. et al. Multiplexed genome regulation in vivo with hyper-efficient Cas12a . Nat. Cell Biol . ( 2022 ) doi: 10.1038/s41556-022-00870-7 . OpenUrl CrossRef PubMed 31. ↵ Wang , D. , Zhang , F. & Gao , G . CRISPR-based therapeutic genome editing: Strategies and in vivo delivery by AAV vectors . Cell 181 , 136 – 150 ( 2020 ). OpenUrl CrossRef PubMed 32. ↵ Zhang , Y. et al. Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system . Sci. Adv . 6 , eaay6812 ( 2020 ). 33. ↵ Replogle , J. M. et al. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors . ( 2022 ) doi: 10.1101/2022.07.13.499814 . OpenUrl Abstract / FREE Full Text 34. ↵ Doench , J. G . Am I ready for CRISPR? A user’s guide to genetic screens . Nat. Rev. Genet . 19 , 67 – 80 ( 2018 ). OpenUrl CrossRef PubMed 35. Bock , C. et al. High-content CRISPR screening . Nature Reviews Methods Primers 2 , 1 – 23 ( 2022 ). OpenUrl CrossRef 36. ↵ Przybyla , L. & Gilbert , L. A . A new era in functional genomics screens . Nat. Rev. Genet . 23 , 89 – 103 ( 2022 ). OpenUrl CrossRef PubMed 37. ↵ Papapetrou , E. P. et al. Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells . Nat. Biotechnol . 29 , 73 – 78 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 38. ↵ Papapetrou , E. P. & Schambach , A . Gene insertion into genomic safe harbors for human gene therapy . Mol. Ther . 24 , 678 – 684 ( 2016 ). OpenUrl CrossRef PubMed 39. ↵ O’Geen , H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression . Nucleic Acids Res . 45 , 9901 – 9916 ( 2017 ). OpenUrl CrossRef PubMed 40. ↵ Karikó , K. , Buckstein , M. , Ni , H. & Weissman , D . Suppression of RNA recognition by Toll- like receptors: the impact of nucleoside modification and the evolutionary origin of RNA . Immunity 23 , 165 – 175 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 41. Baden , L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine . N. Engl. J. Med . 384 , 403 – 416 ( 2021 ). OpenUrl CrossRef PubMed 42. ↵ Gillmore , J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis . N. Engl. J. Med . 385 , 493 – 502 ( 2021 ). OpenUrl CrossRef PubMed 43. ↵ Hou , X. , Zaks , T. , Langer , R. & Dong , Y . Lipid nanoparticles for mRNA delivery . Nat. Rev. Mater . 6 , 1078 – 1094 ( 2021 ). OpenUrl CrossRef PubMed 44. ↵ Gilbert , L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes . Cell 154 , 442 – 451 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 45. Gilbert , L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation . Cell 159 , 647 – 661 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 46. ↵ Mandegar , M. a. , et al. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs . Cell Stem Cell 18 , 541 – 553 ( 2016 ). OpenUrl CrossRef PubMed 47. ↵ Replogle , J. M. et al. Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq . Cell 185 , 2559 – 2575 .e28 ( 2022 ). OpenUrl CrossRef PubMed 48. ↵ Smits , A. H. et al. Biological plasticity rescues target activity in CRISPR knock outs . Nat. Methods 16 , 1087 – 1093 ( 2019 ). OpenUrl CrossRef PubMed 49. ↵ Kumamoto , T. et al. Direct Readout of Neural Stem Cell Transgenesis with an Integration- Coupled Gene Expression Switch . Neuron 107 , 617 – 630 .e6 ( 2020 ). OpenUrl CrossRef PubMed 50. ↵ Sadelain , M. , Papapetrou , E. P. & Bushman , F. D . Safe harbours for the integration of new DNA in the human genome . Nat. Rev. Cancer 12 , 51 – 58 ( 2011 ). OpenUrl PubMed 51. ↵ Kang , K. et al. An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing . J. Anim. Sci. Biotechnol . 10 , 43 ( 2019 ). 52. ↵ Hendel , A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells . Nat. Biotechnol . 33 , 985 – 989 ( 2015 ). OpenUrl CrossRef PubMed 53. Ma , H. et al. CRISPR-Cas9 nuclear dynamics and target recognition in living cells . J. Cell Biol . 214 , 529 – 537 ( 2016 ). OpenUrl Abstract / FREE Full Text 54. ↵ Ma , H. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging . Nat. Methods 15 , 928 – 931 ( 2018 ). OpenUrl CrossRef PubMed 55. ↵ Zhang , X. et al. Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing . Genome Biol . 24 , 145 ( 2023 ). 56. ↵ Liang , R. et al. Prime editing using CRISPR-Cas12a and circular RNAs in human cells . Nat. Biotechnol . 1 – 9 ( 2024 ). 57. ↵ Litke , J. L. & Jaffrey , S. R . Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts . Nat. Biotechnol . 37 , 667 – 675 ( 2019 ). OpenUrl CrossRef PubMed 58. ↵ Schellenberger , V. et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner . Nat. Biotechnol . 27 , 1186 – 1190 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 59. ↵ Nuñez , J. K. et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing . Cell 184 , 2503 – 2519 .e17 ( 2021 ). OpenUrl CrossRef PubMed 60. ↵ Wang , R. N. et al. Bone Morphogenetic Protein (BMP) signaling in development and human diseases . Genes Dis . 1 , 87 – 105 ( 2014 ). OpenUrl CrossRef PubMed 61. ↵ Heldin , C. H. , Miyazono , K. & ten Dijke , P . TGF-beta signalling from cell membrane to nucleus through SMAD proteins . Nature 390 , 465 – 471 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 62. ↵ Massagué , J. TGF-beta signal transduction . Annu. Rev. Biochem. 67 , 753 – 791 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 63. ↵ Korchynskyi , O. & ten Dijke , P . Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter . J. Biol. Chem . 277 , 4883 – 4891 ( 2002 ). OpenUrl Abstract / FREE Full Text 64. ↵ Antebi , Y. E. et al. Combinatorial Signal Perception in the BMP Pathway . Cell 170 , 1184 – 1196 .e24 ( 2017 ). OpenUrl CrossRef PubMed 65. ↵ Perez-Pinera , P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors . Nat. Methods 10 , 239 – 242 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 66. Zhang , Z. , Wu , E. , Qian , Z. & Wu , W.-S . A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets . Sci. Rep . 4 , 7338 ( 2014 ). OpenUrl CrossRef PubMed 67. Beerli , R. R. & Barbas , C. F ., 3rd. Engineering polydactyl zinc-finger transcription factors . Nat. Biotechnol . 20 , 135 – 141 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 68. Fire , A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 391 , 806 – 811 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 69. Paddison , P. J. , Caudy , A. A. & Hannon , G. J . Stable suppression of gene expression by RNAi in mammalian cells . Proc. Natl. Acad. Sci. U. S. A . 99 , 1443 – 1448 ( 2002 ). OpenUrl Abstract / FREE Full Text 70. ↵ Abudayyeh , O. O. et al. C2c2 is a single-component programmable RNA-guided RNA- targeting CRISPR effector . Science 353 , aaf5573 ( 2016 ). 71. ↵ Ecco , G. , Imbeault , M. & Trono , D . KRAB zinc finger proteins . Development 144 , 2719 – 2729 ( 2017 ). OpenUrl Abstract / FREE Full Text 72. ↵ Wang , X. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery . Nat Protoc 18 , 265 – 291 ( 2023 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 16, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation Bo Gu , James M. Linton , Brice Graham Hendrickson , Hengyu Li , Ron Hadas , Gal Manella , Jan Gregrowicz , Michael B. Elowitz bioRxiv 2025.06.15.659618; doi: https://doi.org/10.1101/2025.06.15.659618 Share This Article: Copy Citation Tools Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation Bo Gu , James M. Linton , Brice Graham Hendrickson , Hengyu Li , Ron Hadas , Gal Manella , Jan Gregrowicz , Michael B. Elowitz bioRxiv 2025.06.15.659618; doi: https://doi.org/10.1101/2025.06.15.659618 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Synthetic Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18592) Cell Biology (25507) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24321) Genetics (15610) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17182) Neuroscience (88618) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7641) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)