Programmable RNA writing with trans-splicing

Abstract

RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5' or 3' trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence. Using Cas7-11 cleavage of pre-mRNAs to bias towards editing outcomes, we boost the efficiency of RNA trans-splicing by 10-100 fold, achieving editing rates between 5-50% and 85% on endogenous and reporter transcripts, respectively, while maintaining high-fidelity. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all 12 possible transversions and transitions, insertions ranging from 1 to 1,863 nucleotides, and deletions. We show high efficiency replacement of exon 4 of MECP2, addressing most mutations that drive the Rett Syndrome; editing of SHANK3 transcripts, a gene involved in Autism; and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington's disease. Whole transcriptome sequencing reveals the high precision of PRECISE editing and lack of off-target trans-splicing activity. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing, with demonstrated efficiency in editing HTT exon 1 via AAV delivery. We show that the high activity of PRECISE editing enables editing in non-dividing neurons and patient-derived Huntington's disease fibroblasts. PRECISE editing markedly broadens the scope of genetic editing, is straightforward to deliver over existing gene editing tools like prime editing, lacks permanent off-targets, and can enable any type of genetic edit large or small, including edits not otherwise possible with existing RNA base editors, widening the spectrum of addressable diseases.

Extended Data Figure 1 |. Optimization of PRECISE editing on Gluc reporter transcripts.

(a) Splicing assay employing a split-luciferase reporter reconstituting a Gaussia luciferase transcript through PRECISE RNA editing, contrasting 3′ cargos with and without a branching point (BP), polypyrimidine tract (PPT), or splicing signal (SS). Trans-template contains C-terminal fragments of Gaussia luciferase and a hybridization region. Three DisCas7-11 targeting guides are compared to a non-targeting guide. Gaussia luciferase values normalized to constitutively co-expressed Cypridina luciferase control. (b) Splicing assay employing a split-luciferase reporter reconstituting a Gaussia luciferase transcript through PRECISE RNA editing, contrasting 3′ cargos with and without a branching point (BP), polypyrimidine tract (PPT), or splicing signal (SS). Trans-template contains C-terminal fragments of G-luciferase and cargo hybridization region 2. Three DisCas7-11 targeting guides are compared to a non-targeting guide. Gaussia luciferase values normalized to constitutively co-expressed Cypridina luciferase control. (c) RNA editing rates of the PRECISE editing conditions in part (a) exploring different DisCas7-11 targeting guides and effect of different regulatory signals on the trans-template. (d) RNA editing rates of the PRECISE editing conditions in part (b) exploring different DisCas7-11 targeting guides and effect of different regulatory signals on the trans-template.

Extended Data Figure 2 |. Optimization of PRECISE editing trans-templates on endogenous transcripts.

(a) RNA editing rate of PRECISE on the endogenous STAT3 transcript using trans-templates with different cargo guide lengths and positions and either active DisCas7-11 or dDisCas7-11. (b) RNA editing rate of PRECISE on the endogenous PPIB transcript using trans-templates with different cargo guide lengths and positions and either active DisCas7-11 or dDisCas7-11. (c) RNA editing rate of PRECISE on the endogenous STAT3 transcript using trans-templates with different hybridization lengths and extensions. (d) RNA editing rate of PRECISE on the endogenous PPIB transcript using trans-templates with different hybridization lengths and extensions. (e) RNA editing rate of PRECISE on the endogenous STAT3 transcript using trans-templates with different linker lengths between the cargo guide and the splicing sequence. (f) RNA editing rate of PRECISE on the endogenous PPIB transcript using trans-templates with different linker lengths between the cargo guide and the splicing sequence.

Extended Data Figure 3 |. Comprehensive DisCas7-11 guide and cargo guide tiling for optimizing PRECISE editing efficiency.

(a) RNA editing rate of PRECISE on the endogenous STAT3 transcript using trans-templates with different DisCas7-11 guides paired with different cargo guides and either active DisCas7-11 or dDisCas7-11. (b) RNA editing rate of PRECISE on the endogenous PPIB transcript using trans-templates with different DisCas7-11 guides paired with different cargo guides and either active DisCas7-11 or dDisCas7-11. (c) Evaluation of high-fidelity Cas13d (hfCas13d) for trans-splicing at the PPIB transcript compared to PRECISE editing with DisCas7-11. A panel of targeting guides is compared to a non-targeting guide for each of the nucleases, as indicated.

Extended Data Figure 4 |. Exploration of Cas7-11 orthologs and their application to PRECISE editing.

(a) A panel of Cas7-11 orthologs is tested with every known Cas7-11 ortholog crRNA in either the sense or antisense direction for knockdown of G-luciferase activity in HEK293FT cells. (b) Top four Cas7-11 orthologs are evaluated for their ability to stimulate 3′ PRECISE editing on the endogenous SHANK3 transcript. Each ortholog is evaluated with DRs from other orthologs that maximally enabled knockdown in part (a). Orthologs are tested with either the full length DRs or predicted mature DRs. (c) Top four Cas7-11 orthologs are evaluated for their ability to stimulate 5′ PRECISE editing on the endogenous HTT transcript. Each ortholog is evaluated with DRs from other orthologs that maximally enabled knockdown in part (a). Orthologs are tested with either the full length DRs or predicted mature DRs. Hsm, “Hydrothermal sediment microbial communities from Guaymas Basin, California, USA 4484” Cas7-11; Hvs, “Hydrothermal vent sediment bacterial communities from Southern Trench, Guaymas Basin, Mexico-4870-07-3-4_MG” Cas7-11.

Extended Data Figure 5 |. Mutagenesis of DisCas7-11 for improved catalytic activity and higher trans-splicing rates via PRECISE editing.

(a) Panel of DisCas7-11 single protein mutants evaluated for G-luciferase knockdown. G-luciferase activity is normalized to a C-luciferase control and knockdown activity is calculated relative to a non-targeting guide. (b) Panel of DisCas7-11 single protein mutants evaluated for endogenous STAT3 3′ PRECISE editing. A targeting guide is compared to a non-targeting guide. (c) Panel of DisCas7-11 single protein mutants evaluated for endogenous PPIB 3′ PRECISE editing. A targeting guide is compared to a non-targeting guide.

Extended Data Figure 6 |. Engineering of trans-template cargos for efficient PRECISE editing.

(a) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous PPIB transcript with trans-template cargos carrying different branch point sequences. Editing is compared between targeting and non-targeting crRNAs. (b) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous STAT3 transcript with trans-template cargos carrying different branch point sequences. Editing is compared between targeting and non-targeting crRNAs. (c) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous PPIB transcript with trans-template cargos carrying shorter branch point sequence variants. Editing is compared between targeting and non-targeting crRNAs. (d) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous PPIB transcript with trans-template cargos carrying different intron splicing enhancer (ISE) sequences. Editing is compared between eDisCas7-11 and dDisCas7-11. (e) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous STAT3 transcript with trans-template cargos carrying different ISE sequences. Editing is compared between eDisCas7-11 and dDisCas7-11. (f) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous STAT3 transcript with trans-template cargos carrying different exon splicing enhancer (ESE) sequences. Editing is compared between targeting and non-targeting guides. (g) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous SHANK3 transcript with trans-template cargos carrying different exon splicing enhancer (ESE) sequences. Editing is compared between targeting and non-targeting guides. (h) Evaluation of 3′ trans-splicing PRECISE editing on the endogenous STAT3 transcript with trans-template cargos carrying different nuclear retention element (NRE) sequences. Editing is compared between targeting and non-targeting guides.

Extended Data Figure 7 |. Enhanced trans-splicing efficiencies with splicing factor recruitment and further characterization of edit types possible with PRECISE editing.

(a) Evaluation of 3′ trans-splicing PRECISE editing on endogenous STAT3 transcript with DisCas7-11 fusions to different splicing factors. Trans-splicing with a targeting DisCas7-11 guide is compared to a non-targeting guide. Fusions are on either the N- or C-terminus and either an XTEN linker or no linker is used. (b) Evaluation of 3′ trans-splicing PRECISE editing on endogenous PPIB transcript with DisCas7-11 fusions to different splicing factors. Trans-splicing with a targeting DisCas7-11 guide is compared to a non-targeting guide. Fusions are on either the N- or C-terminus and either an XTEN linker or no linker is used. (c) Evaluation of 3′ trans-splicing PRECISE editing on endogenous TOP2A transcript with DisCas7-11 fusions to different splicing factors. Trans-splicing with a targeting DisCas7-11 guide is compared to a non-targeting guide. Fusions are on either the N- or C-terminus and either an XTEN linker or no linker is used. (d) Evaluation of 3′ trans-splicing PRECISE editing on endogenous PABPC1 transcript with DisCas7-11 fusions to different splicing factors. Trans-splicing with a targeting DisCas7-11 guide is compared to a non-targeting guide. Fusions are on either the N- or C-terminus and either an XTEN linker or no linker is used. (e) Heatmap of 3’ PRECISE editing efficiency on 4 endogenous targets, PPIB, STAT3, TOP2A, and PABPC1. Fusions are on either the N- or C-terminus and either an XTEN linker or no linker is used. Rates are expressed as fold changes relative to DisCas7-11 with no fusion. (f) Evaluation of 3′ trans-splicing PRECISE editing on endogenous STAT3 transcript for trans-templates carrying different types of edits, including amino acid mutations, frameshifts, and nucleotide insertions. (g) Evaluation of 3′ trans-splicing PRECISE editing on endogenous PPIB transcript for trans-templates carrying different amino acid mutation edits. (h) Evaluation of 3′ trans-splicing PRECISE editing on endogenous PPIB transcript for trans-templates carrying different edits, including frameshifts, nucleotide insertions, and nucleotide deletions.

Extended Data Figure 8 |. Exploration of transcript perturbations and off-targets due to PRECISE editing.

(a) Relative expression of four different endogenous transcripts targeted with DisCas7-11 PRECISE editing, as measured by qPCR. For each replicate, transcript expression is normalized to GAPDH expression and then adjusted transcript expression is normalized to a DisCas7-11 non-targeting guide control. Conditions shown include PRECISE editing with dDisCas7-11 (D), active DisCas7-11 (WT), and eDisCas7-11 (E). (b) Differentially expressed genes for a set of pairwise comparisons from whole-transcriptome RNASeq performed on PPIB-targeting PRECISE edited HEK293FT cells. Numbers of upregulated genes, downregulated genes, and total counts for each comparison are shown. T and NT indicate targeting or non-targeting guides, respectively. (+) and (d) indicate wild-type DisCas7-11 and dDisCas7-11, respectively. (c) Evaluation of 3′ PRECISE editing on the endogenous PPIB transcript for samples used for whole transcriptome RNA sequencing. T and NT indicate targeting or non-targeting guides, respectively. (+) and (d) indicate wild-type DisCas7-11 and dDisCas7-11, respectively. (d) Adaptation of Integrative Genomics Viewer plot of the endogenous PPIB transcript subjected to PRECISE editing, mapping whole-transcriptome RNAseq reads.

Extended Data Figure 9 |. Internal trans-splicing by PRECISE using split-luciferase reporter.

(a) Schematic of internal exon PRECISE editing showing replacement of G-luciferase exons in a reporter to restore luciferase activity. Approximate positions are shown for both 5′ and 3′ tiling positions of crRNAs and tgRNAs. (b) Evaluation of insertion-spanning internal PRECISE editing on overexpressed luciferase splicing reporter reconstituting functional G-luciferase, measured by NGS. Cargos with tgRNAs targeted 5′ and 3′ to the inserted exons or scrambled guide sequences are compared with either active or dDisCas7-11 with different pairs of crRNAs. Different combinations of 5′ and 3′ intron targeting PRECISE guides are shown. NT (cargo tgRNAs) denotes scrambled tgRNA guide sequence. NT (crRNA) denotes a non-targeting guide for DisCas7-11.

Extended Data Figure 10 |. Additional characterization of 5′ trans-splicing with PRECISE editing.

(a) Evaluation of 5′ PRECISE editing on endogenous HTT transcript in iPSC-derived human neurons by AAV delivery of protein-free cargo with HDV ribozyme. 3 different doses of AAV for the delivery of a targeting cargo or a control GFP vector are compared. (b) Evaluation of 5′ PRECISE editing on endogenous HTT transcripts with wild-type small DisCas7-11 and mutant versions. A targeting guide is compared to a non-targeting guide. (c) Evaluation of 3′ PRECISE editing on endogenous SHANK3 transcripts with wild-type small DisCas7-11 and mutant versions. A targeting guide is compared to a non-targeting guide. (d) Demonstration of 5′ PRECISE editing of the HTT transcript with lentiviral delivery of DisCas7-11, guide, and RNA trans-template in HEK293FT cells. RNA writing into the endogenous HTT transcript with targeting and non-targeting DisCas7-11 guides is compared.

Figure 1 |. Development of PRECISE RNA editing.

(a) Schematic showing 3′ PRECISE editing with a trans-splicing cargo and Cas7-11 crRNA targeted to a 3′ region of the intron. BP, branch point; PPT, polypyrimidine tract. (b) Splicing assay employing a split-luciferase reporter reconstituting a Gaussia luciferase transcript through PRECISE RNA editing, contrasting 3′ cargos with and without a branching point (BP), polypyrimidine tract (PPT), or splicing signal (SS). A DisCas7-11 targeting guide is compared to a non-targeting guide. Gaussia luciferase values normalized to a constitutively co-expressed Cypridina luciferase control. (c) RNA editing analysis of the splicing assay, using a barcoded cargo and sequencing primers spanning the splice junction. (d) Splicing assay employing a split-luciferase reporter reconstituting a Gaussia luciferase transcript through PRECISE RNA editing demonstrating three different DisCas7-11 targeting guides compared against a non-targeting guide and two different cargo hybridization guides (1 and 2). Gaussia luciferase values normalized to constitutively co-expressed Cypridina luciferase control. Wt denotes wild-type DisCas7-11. Dead denotes catalytically inactive DisCas7-11. (e) RNA editing analysis for the splicing assay, using a barcoded cargo and sequencing primers spanning the splice junction. (f) Endogenous PRECISE editing for a set of crossed guides and cargos targeting the STAT3 pre-mRNA. Editing rates expressed as % of reads containing “barcoding” mutations / total reads of “barcoded” and wild-type transcripts. NT guide, scrambled crRNA; NT cargo, scrambled cargo guide. (g) Endogenous PRECISE editing for a set of crossed guides and cargos targeting the PPIB pre-mRNA. Editing rates expressed as % of reads containing “barcoding” mutations / total reads of “barcoded” and wildtype transcripts. NT guide, scrambled crRNA; NT cargo, scrambled hybridization. (h) Comparison of splicing rates for a set of cargos targeting STAT3 with either Cas7-11 or Cas13 crRNAs targeting the same STAT3 pre-mRNA intron. Psp, Prevotella sp. Cas13; Lwa, Leptotrichia wadei Cas13; Ruminococcus flavifaciens Cas13. All constructs are cloned with 5′ and 3′ flanking SV40 bipartite NLS sequences.

Figure 2 |. Rational engineering of DisCas7-11 catalytic activity to improve PRECISE editing.

(a) Structure of DisCas7-11 complexed with a crRNA (light blue) and tgRNA (dark blue) showing the positions of residues targeted for mutagenesis for improving RNase activity. Residues with mutations causing a large increase in cleavage efficiency are highlighted (yellow). (b) Zooms showing the positions of residues selected for mutagenesis to generate enhanced Cas7-11 catalytic variants. (c) Knockdown efficiency of a panel of single, double, and triple mutants of DisCas7-11 expressed as a ratio between Gaussia (targeted) and Cypridina luciferase (constitutive) expression, normalized to the ratio for a non-targeting guide condition. The dotted line indicates Gaussia luciferase expression levels with knockdown by wild-type DisCas7-11. The knockdown efficiency as a percentage is denoted above select conditions. (d) Schematic showing the construction of a splicing reporter for PRECISE editing, showing the N-terminal domain of Gaussia luciferase (blue) in exon 1 with the intron 46 from COL7A1 gene. The Gluc pre-mRNA is expressed and the PRECISE cargo (blue) is shown bound to the intron, carrying the remaining C-terminal sequence of Gaussia luciferase in another exon. (e) Splicing assay employing a split-luciferase reporter reconstituting a Gaussia luciferase transcript through PRECISE, using two cargos with different cargo hybridization guides and either catalytically inactive (dDisCas7-11), wild-type DisCas7-11 (wtDisCas7-11), or a high-performing triple mutant of Cas7-11 (DisCas7-11^{Y312K/D988K/D1580R}). Gaussia luciferaserelative expression is quantified as Gaussia luciferase expression normalized to Cypridina luciferase expression. Fold changes are relative to rates with dDisCas7-11. (f) Schematic showing the exons and introns targeted for endogenous PRECISE editing for each endogenous transcript evaluated. Only flanking exons are shown with position indicated by number. Flanking exon and targeted intron sizes and cargo and guide positions are to scale. Transcripts per million expression levels for the targeted genes in HEK293FT cells are plotted. (g) PRECISE RNA editing quantified by next-generation sequencing for a panel of endogenous targets at the positions indicated in (f) with different DisCas7-11 mutants. Percent editing represents the ratio between junction reads containing barcoded silent mutations to total reads of the targeted exon-exon boundary.

Figure 3 |. Optimization and evaluation of PRECISE editing capabilities, including small and large edits, insertions, and multiplexing.

(a) Schematic showing the 3′ trans-splicing approach for optimization of PRECISE template branch points, intronic splicing enhancers, exonic splicing enhancers, and nuclear retention elements. (b) Evaluation of a panel of different branch point sequences for PRECISE 3′ trans-splicing editing of the endogenous STAT3 transcript. (c) Evaluation of a panel of different branch point sequences for PRECISE 3′ trans-splicing editing of the endogenous PPIB transcript. (d) Installation of all possible single point mutations and other changes at the STAT3 endogenous transcript exon 6 with PRECISE editing. Editing is compared with targeting and non-targeting DisCas7-11 guides. (e) Installation of all possible single point mutations and other changes at the PPIB endogenous transcript exon 5 with PRECISE editing. Editing is compared with targeting and non-targeting DisCas7-11 guides. (f) Installation of varying sized inserts at the STAT3 endogenous transcript exon 6 with PRECISE editing. Editing is compared with targeting and non-targeting DisCas7-11 guides. (g) Multiplexed PRECISE editing of 81–120 bp sized inserts at five different endogenous transcripts: STAT3 (S), PPIB (P), USF1 (U), PABPC1 (P), and TOP2A (T). Editing is measured via next-generation sequencing.

Figure 4 |. Evaluation of protein level edits, transcriptome-wide specificity, and delivery by lentivirus.

(a) Evaluation of USF1 protein editing due to PRECISE-based RNA writing by Western Blotting. USF1 is overexpressed and is targeted with either targeting or non-targeting DisCas7-11 guides and targeting or non-targeting cargo hybridization guides. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (b) Evaluation of endogenous HDAC1 protein editing due to PRECISE-based RNA editing by Western Blotting. The HDAC1 transcript is targeted with either targeting or non-targeting DisCas7-11 guides and targeting or non-targeting cargo hybridization guides. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (c) Evaluation of endogenous PPIB protein editing due to PRECISE-based RNA writing by Western Blotting. The PPIB transcript is targeted with either targeting or non-targeting DisCas7-11 guides and a targeting cargo hybridization guide. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (d) Volcano plots of differentially expressed genes as measured by transcriptome-wide sequencing of cells with PPIB PRECISE editing. (e) Number of significant trans-spliced off-targets identified by transcriptome-wide sequencing of cells following PRECISE editing of PPIB. (f) Schematic of PRECISE RNA writing for 3′ trans-splicing via lentiviral delivery. Shown are the construct designs for a two-vector system. (g) Demonstration of PRECISE editing of the SHANK3 transcript with lentiviral delivery of DisCas7-11 expression, guides, and RNA trans-template in HEK293FT cells. RNA writing of a 108 bp sequence into the endogenous SHANK3 transcript with targeting and non-targeting DisCas7-11 guides is compared.

Figure 5 |. Development of PRECISE editing for efficient 5′ trans-splicing without proteins and only using the RNA trans-splicing template.

(a) Schematic showing 5′ PRECISE with two different approaches: 1) Pre-mRNA targeting is shown with a trans-splicing cargo and DisCas7-11 crRNA targeted to a 3′ region of the intron area targeted by the cargo hybridization guide; and 2) Cargo targeting is shown where the DisCas7-11 guide cleaves the 3′ end of the trans-template, separating the poly(A) tail from the cargo. (b) RNA editing rates for using PRECISE editing to replace HTT exon 1 with a short synthetic exon using DisCas7-11 constructs that either target the pre-mRNA intron or the cargo template. Active DisCas7-11 is compared with dDisCas7-11. (c) RNA editing rates using PRECISE to replace HTT exon 1 with a short synthetic first exon. Rates are compared for constructs with different combinations of regulatory sequences, including intronic splicing enhancers (ISEs), branch points (BPs), and splicing signals. Cargos with either no 5′ consensus splicing signal, indicated as (/), or a GUGAGU or GUAAGU sequence are compared. (d) RNA editing rates for 5′ trans-splicing PRECISE editing on the endogenous PPIB transcript using a set of cargos with hybridization guides targeting different regions of PPIB intron 4. A DisCas7-11 crRNA targeting the 3′ end of the cargo payload is compared with a non-targeting DisCas7-11 guide. (e) RNA editing rates for 5′ trans-splicing PRECISE editing on the endogenous PABPC1 transcript using a set of cargos with hybridization guides targeting different regions of PABPC1 intron 13. A targeting DisCas7-11 crRNA targeting the 3′ end of the cargo payload is compared with a non-targeting DisCas7-11 guide (f) RNA editing rates for 5′ trans-splicing PRECISE editing on the endogenous RPL41 transcript using a set of cargos with hybridization guides targeting different regions of RPL41 intron 2. A targeting DisCas7-11 crRNA targeting the 3′ end of the cargo payload is compared with a non-targeting DisCas7-11 guide. (g) Schematic of protein-free PRECISE editing using ribozymes to liberate trans-templates of the poly(A) tail and enable trans-splicing based RNA writing. (h) RNA editing rate of 5′ trans-splicing PRECISE editing on the endogenous HTT transcript with trans-templates cut by either DisCas7-11 or one of several ribozymes. DisCas7-11 targeting is compared to a non-targeting guide. (i) RNA editing rate of 5′ trans-splicing PRECISE editing on the endogenous RPL41 transcript with trans-templates cut by either DisCas7-11 or one of several ribozymes. DisCas7-11 targeting is compared to a non-targeting guide. (j) RNA editing rate of 5’ PRECISE editing on the endogenous HTT transcript in HEK-293FT cells by AAV delivery. Both targeting cargo guides and non-targeting cargo guides are compared using AAV delivery. (k) RNA editing rate of 5′ trans-splicing PRECISE editing on the endogenous HTT transcript in expanded-repeat patient-derived fibroblasts via lentiviral delivery. Trans-templates are cut by DisCas7-11 using a two-vector system at different titer amounts. (l) RNA editing rate of 5′ trans-splicing PRECISE editing on the endogenous HTT transcript in iPSC-derived human neurons via lentiviral delivery. Trans-templates are cut by DisCas7-11 using a two-vector system at different titer amounts.