CRISPR-CAS12A DIRECTED RANDOM MUTAGENESIS AGENTS AND METHODS

Abstract

Disclosed are new nucleic acid base-editing systems comprising fusion proteins comprising a) an RNA-programmable nucleic acid recognition module or other suitable nucleic acid recognition module, b) a light inducible reactive oxygen generator. Further disclosed are methods and kits to modify or mutagenize a target DNA region in prokaryotic or eukaryotic cells or organisms.

Claims

1. A method for inducing one or more modifications in a target nucleic acid molecule, comprising the steps: a) contacting the target nucleic acid molecule with a fusion protein comprising: i) a nucleic acid recognition module (NARM); ii) a protein that generates reactive oxygen species (ROSP); iii) an optional a linker peptide between the NARM and the ROSP; b) in the presence of a guide RNA complementary to one strand of the target nucleic acid molecule, and c) an activation of the ROSP.

2. A method according to claim 1, wherein the NARM is a catalytically inactive guided-nuclease, and in the presence of a guide RNA complementary to one strand of the target nucleic acid molecule, and an activation of the ROSP by illumination with light of an appropriate wavelength.

3. A method according to claim 2, wherein the NARM is a catalytically inactive guided-nuclease, selected from Cas12a, preferably dLbaCpf1, and the ROSP is selected from mOrange2 or Pp2FbFP_L30M.

4. A method according to claim 1, wherein the i) NARM is dLbaCpf1, ii) ROSP is mOrange2 fluorescent protein or Pp2FbFP_L30M and in the presence of a guide RNA complementary to one strand of the target nucleic acid molecule, and an activation of the ROSP by illumination with light of an appropriate wavelength to obtain a sufficient excitation of the ROSP.

5. A method according to any of the claims 1 to 4, wherein the modification is carried out in a prokaryotic or eukaryotic cell.

6. A method according to any of the claims 1 to 5, wherein the modification is carried out in a plant.

7. A method according to claim 5, wherein the wherein the modification is carried out in a prokaryotic cell.

8. A method according to claim 7, wherein the wherein the prokaryotic cell is of the genus Bacillus.

9. A method according to claim 7, wherein the wherein the prokaryotic cell is a strain of Bacillus subtilis.

10. A recombinant protein comprising i) dLbaCpf1; ii) mOrange2 fluorescent protein or Pp2FbFP_L30M.

11. A system comprising: (i) a fusion protein according to any of the claims 1 to 4, and 9, (ii) a guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the components (i) and (ii) are cloned into a appropriate plasmid which allows expression in the host cell.

Description

EXAMPLES

Example 1: Bacillus Subtilis Upp/5-FU Assay System

[0137] Described herein is the Bacillus subtilis upp gene and knockout mutations resulting in resistance to 5-fluorouracil (5-FU) as a system to characterize mutation rates and mutation types induced by random mutagenesis, according to the invention.

[0138] The B. subtilis (substrain 168) upp gene (NCBI Reference Sequence: NC_000964.3, https://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3; as retrieved on May 29, 2020) encodes an uracil phosphoribosyltransferase enzyme which is a pyrimidine salvage enzyme. This enzyme also converts 5-fluorouracil directly to 5-fluorouridine monophosphate, a very potent inhibitor of thymidylate synthetase (Neuhard, 1983). As a result, culturing B. subtilis on plates supplemented with 5-FU causes toxicity for all cells expressing a functioning upp gene and selection for cells lacking a functional copy (Fabret, 2002). This makes upp a useful target for detecting very low levels of mutagenesis, with a variety of potential mutations (e.g., additions, deletions, substitutions) that will result in selectable loss-of-function. The small size of this gene (630 bp, 210 aa) allows for PCR amplicon sequencing and rapid analysis of numerous potential mutations. It should be noted that in the upp gene is inessential when ample uracil is supplied.

[0139] ROS induced mutations at upp: Reactive oxygen species vary in their reactivity and their mechanism of mutagenesis. Singlet oxygen has been demonstrated to induce a broad spectrum of mutations including single-nucleotide variants and chromosomal deletions (Noma, 2012). The most frequent product of oxidative damage to DNA is 8-oxo-2′-deoxyguanosine, which preferentially pairs with adenine rather than cytosine, resulting in G:C.fwdarw.T:A base pair transversions. Any out-of-frame insertion/deletion mutation in the first 441 nt of the upp gene will result in a premature stop before the C-terminal active site that would result in upp loss-of-function. There are 21 potential G:C.fwdarw.T:A base pair transversions that would result in a premature stop before the C-terminal active site (provided in Table 1).

TABLE-US-00001 TABLE 1 Predicted G:C.fwdarw.T:A transversions within the first 486 nt of upp resulting in premature stop. Resulting Stop Codon WT Codon, AA (n) TGA GGA, G (5) TGC, C (0) TAA GAA, E (10) TCA, S (1) TAC, Y (2) TAG GAG, E (2) TCG, S (1)

[0140] In a preliminary sequence analysis of 94 selected 5-FU-resistant mutants, 68 of the sequenced mutants (72%) exhibited G:C.fwdarw.T:A base pair transversions.

Example 2: Generating Guided ROSProduction Strains of B. subtilis Str 168

[0141] Identified upp target sites for the RNA-guided DNA-binding protein dLbaCpf1: Five guide sequences were chosen to target dLbaCpf1 to the upp gene of B. subtilis. As off-target controls, five guide sequences were chosen to target the amyE gene of B. subtilis (NCBI Reference Sequence: NC_000964.3, https://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3). For both sets, target sequences were chosen for their predicted score using DeepCpf1 (Kim, 2018). Also, targets were designed to eliminate effects of CRISPR-induced inhibition (CRISPRi) by targeting the nontemplate strand (Seong Keun Kim, Haseong Kim, Woo-Chan Ahn, Kwang-Hyun Park, Eui-Jeon Woo, Dae-Hee Lee, and Seung-Goo Lee, ACS Synthetic Biology 2017 6 (7), 1273-1282, DOI: 10.1021/acssynbio.6b00368).

[0142] Guide expression constructs: An array of guide expression vectors was constructed with a synthetic, broad-spectrum constitutive promoter driving a series of direct repeats and 23-bp targeting sequences terminated by a T7 terminator.

[0143] All guide expression cassettes were inserted into pBV70, a modified derivative of pMiniMAD2 (Patrick & Kearns, 2008) between BamHI and EcoRI restriction sites. These plasmids included selectable markers conferring resistance to the antibiotics ampicillin (for E. coli cloning) and erythromycin (for B. subtilis maintenance), origins from pBR322 (for E. coli cloning) and temperature-sensitive pE194 (for B. subtilis maintenance), and a mobilization fragment (mob) to allow bacterial conjugation.

[0144] ROSProducing Cas-protein fusion constructs: An array of dLbaCpf1 expression plasmids were constructed with different ROSProducing fusions, connected by a short flexible linker. These fusion products (and one plasmid without a fused protein) included nuclear localization sequences at either end and were driven by a synthetic, broad-spectrum constitutive promoter and terminated by a T7 terminator.

[0145] All dLbCpf1 expression plasmids included selectable markers conferring resistance to the antibiotic kanamycin, and origins from pBR322 (for E. coli cloning) and temperature-sensitive pE194 (for B. subtilis maintenance).

Example 3: Guided ROS Mutagenesis of Upp Gene in the Presence of Catalytically Inactive RNA-Guided Endonucleases and Upp Guide RNAs

[0146] To test the rate and spectrum of mutations induced by guided ROSProduction, an experiment was performed where B. subtilis str. 168 cells were co-transformed with combinations of guide expression and fusion dLbaCpf1 expression plasmids (Table 2).

TABLE-US-00002 TABLE 2 Combinations of guide expression and fusion dLbaCpf1 expression plasmids used for this experiment. Strain SEQ ID Guide SEQ ID Name Fusion dLbaCpf1 plasmid NO: Plasmid NO: 0353 pBV003 3 pBV053 5 (dLbaCpf1-mOrange2) (3X upp) 3341 pBV033 4 pBV041 6 (dLbaCpf1-Pp2FbFP_L30M) (amyE) 3353 pBV033 4 pBV053 5 (dLbaCpf1-Pp2FbFP_L30M) (3X upp)

[0147] Light-induced mutagenesis treatment: A single colony for each plasmid combination was inoculated into LB medium supplemented with lincomycin (25 mg/L), kanamycin (5 mg/L) and erythromycin (1 mg/L) and cultured overnight at 30° C. Overnight cultures were diluted 25-fold into fresh selective media and arrayed into 24-well blocks for incubation at 30° C. with agitation and with or without illumination cycling (15 min on, 1 hr off) over 24 hours.

[0148] Determining 5-FU resistant counts: Cultures were diluted 10-fold into LB and 100 μL were plated in triplicate onto LB agar plates containing 6.5 mg/L 5-FU to quantify resistant CFU. After a 24-hour incubation at 37° C., resistant CFU were counted.

[0149] To determine total viable count, treated cultures were further diluted to 10.sup.5 in LB and plated onto LB agar plates to quantify total CFU. After an overnight incubation at 30° C., total CFU were counted. The results of this experiment are demonstrated by rate of resistant CFU in FIG. 1. In this experiment, targeting the upp gene resulted in a 27-fold increase in rate of resistant CFU relative to targeting amyE gene. Fusion to Pp2FbFP_L30M resulted in an 11-fold increase in rate of resistant CFU relative to mOrange2 fusion. Light cycling resulted in a 22-fold increase in rate of resistant CFU relative to dark treatment.

Example 4: Characterizing the Position and Frequency of Mutations Induced by Guided ROSProduction

[0150] To understand the types of mutations introduced by guided ROSProduction, the upp regions of 5-FU-resistant colonies were amplified by PCR and sequenced.

[0151] Mutational analysis by Sanger sequencing: In one experiment, a guide expression construct containing an array of three guides targeting the B. subtilis upp gene (SEQ ID NO: 5) was transformed into B. subtilis str. 168 harboring a plasmid expressing dead LbaCpf1 fused to either mOrange2 or Pp2FbFP_L30M (SEQ ID NOs: 3, 4). A single colony for each was inoculated into LB medium supplemented with lincomycin (25 mg/L), kanamycin (5 mg/L) and erythromycin (1 mg/L) and cultured overnight at 30° C. Overnight cultures were diluted 25-fold into fresh selective media and arrayed into 24-well blocks for incubation at 30° C. with agitation and illumination cycling (15 min on, 1 hr off) over 24 hours. Cultures were diluted 10-fold into LB and 100 μL were plated in triplicate onto LB agar plates containing 6.5 mg/L 5-FU. After a 24-hour incubation at 37° C., colonies were picked into 150 μL of sterile water and microwaved for 4 minutes to lyse the cells. The upp region was amplified by PCR (SEQ ID NOs: 7, 8) and sequenced using nested primers (SEQ ID NOs: 9, 10). The upp gene was sequenced for 27 colonies with mOrange2 and 57 colonies with Pp2FbFP_L30M. The results of the sequencing analysis are provided below in Table 3.

[0152] Observed mutations to upp in B. subtilis resulting in functional knockout and resistance to 5-FU. Positions are provided with reference to the upp coding sequence.

TABLE-US-00003 TABLE 3 Results of sequencing analysis 590- Position 247 401 404 540 545 547 548 556 579 671 mOrange2 G:C .fwdarw. A:T 0 0 1 0 0 0 0 0 0 0 A:T .fwdarw. G:C 0 1 0 0 0 0 0 0 0 0 G:C .fwdarw. T:A 0 0 1 12 6 0 0 1 0 0 C .Math. G 0 0 0 2 0 1 0 0 0 0 T .Math. A 0 0 0 0 0 0 0 0 1 0 DEL 0 0 0 0 0 0 0 0 0 1 Pp2FbFP_L30M G:C .fwdarw. A:T 1 0 0 0 0 0 0 0 0 0 A:T .fwdarw. G:C 0 0 0 0 0 0 0 0 0 0 G:C .fwdarw. T:A 0 0 0 21 30 0 0 1 0 0 C .Math. G 0 0 0 3 0 0 1 0 0 0 T .Math. A 0 0 0 0 0 0 0 0 0 0 DEL 0 0 0 0 0 0 0 0 0 0

[0153] More than 90% of the 57 sequenced upp fragments from 5-FU-resistant colonies generated in Pp2FbFP_L30M fusion strains had G:C.fwdarw.T:A transversion mutations present. These caused premature stops (C540A and G556T) and an A.fwdarw.E substitution (C554A).

Example 5: Testing Several Potential ROSProducing Proteins for Guided Upp Mutagenesis

[0154] An experiment was designed to test different potential ROSProducing proteins for their ability to deliver guided mutagenesis. Tested proteins included fluorescent proteins (SuperNova, tagRFP, mOrange2) and flavin-mononucleotide-binding (Fb) proteins (SOPP3, Pp2FbFP_L30M, and an experimental chimera of the two). The variety of chosen proteins was predicted to produce differing levels of superoxide and singlet oxygen reactive oxygen species, with different photon efficiencies.

TABLE-US-00004 TABLE 4 dLbaCpf1 fusion expression vectors generated to test the mutagenic effects of different ROS producers. Fusion Protein SEQ ID NO: mOrange2 3 tagRFP 11 SuperNova 12 SOPP3 13 Pp2FbFP_L30M 4 Chimera 14

[0155] This experiment was completed for mOrange2, tagRFP, SOPP3, and Pp2FbFP_L30M, and the results are in FIG. 2.

[0156] Total viable CFU and resistant CFU counts for light and dark treatments of each plasmid combination. Resistant CFU relative to total CFU were calculated, and the standard deviation of triplicate plates is provided.

[0157] This experiment was completed for the FbFP chimera, SuperNova, Pp2FbFP-L30M, and Pp2FbFP_L30M with guide targeting amyE as a control.

[0158] Total viable CFU and resistant CFU counts for light and dark treatments of each plasmid combination. Resistant CFU relative to total CFU were calculated, and the standard deviation of triplicate plates is provided in FIG. 3.

Example 6: Testing the Effect of Multiple Guides on Increasing ROS-Induced Mutagenesis

[0159] Sequence analysis of 5-FU-resistant strains was using three different guides directed to upp suggested that most mutations occurred at or near one target more frequently than the other two target sites. We designed an experiment to determine if the three guides together worked synergistically to generate greater localized mutagenesis than individually combined.

[0160] Generating guide expression constructs to test individual guide sequences and their combinations: We constructed several guide expression vectors to express guides for this experiment (Table 5).

TABLE-US-00005 TABLE 5 Guide expression vectors generated to test the synergistic effect of multiple proximal guides. Guide(s) SEQ ID NO: amyE1 15 amyE2 6 upp1 16 upp2 17 upp3 18 3X upp 5

[0161] All guide constructs were tested in combination with the dLbaCpf1-Pp2FbFP_L30M expression construct. A single colony for each plasmid combination was inoculated into LB medium supplemented with lincomycin (25 mg/L), kanamycin (5 mg/L) and erythromycin (1 mg/L) and cultured overnight at 30° C. Overnight cultures were diluted 25-fold into fresh selective media and arrayed into 24-well blocks for incubation at 30° C. with agitation and with or without illumination cycling (15 min on, 1 hr off) over 24 hours. Cultures were diluted 10-fold into LB and 100 μL were plated in triplicate onto LB agar plates containing 6.5 mg/L 5-FU to quantify resistant CFU. After a 24-hour incubation at 37° C., resistant CFU were counted. To determine total viable count, treated cultures were further diluted to 10.sup.5 in LB and plated onto LB agar plates to quantify total CFU. After an overnight incubation at 30° C., total CFU were counted.

[0162] The resulting chart summarizing the rate of resistant CFU is provided in FIG. 4. In this experiment, targeting the upp gene with three guides resulted in a 1.8-fold increase in rate of resistant CFU relative to the sum of the rates of all three individually expressed guides. When resistance rate was calculated as increase relative to dark controls (rate in light/rate in dark−1), the three guides resulted in an 3.1-fold increase compared to the sum of the increases from all three individually expressed guides.

Example 7: Deep Amplicon Sequencing of Guided ROS-Induced Mutagenesis

[0163] Amplicon sequencing allows for useful read coverage of very many pooled mutants. We will generate a series of pools of both 5-FU-resistant mutants and unselected culture over repeated 24-hour light cycling experiments with dilution into selective LB for each round. We will test the strains shown in Table 6.

TABLE-US-00006 TABLE 6 The repeated exposure series will be performed using the listed strains. Strain Fusion Guides WT NA NA Un-fused None 5X upp (SEQ ID NO: 19) mOrange2 mOrange2 5X upp Pp2FbFP_L30M Pp2FbFP_L30M 5X upp Non-targeted Pp2FbFP_L30M 5X amyE (SEQ ID NO: 20)

[0164] A single colony for each plasmid combination was inoculated into LB medium supplemented with lincomycin (25 mg/L), kanamycin (5 mg/L) and erythromycin (1 mg/L) and cultured overnight at 30° C. Overnight cultures were diluted 25-fold into fresh selective media and arrayed into 24-well blocks for incubation at 30° C. with agitation and with or without illumination cycling (15 min on, 1 hr off) over 24 hours.

[0165] Sequential light cycling treatments to increase rate of guided ROS-induced mutagenesis: After each 24-hour round of light cycling treatment, 50 μL from each well is used to inoculate 950 μL of LB supplemented with lincomycin (25 mg/L), kanamycin (5 mg/L) and erythromycin (1 mg/L) for another round of light cycling. Also after each round, 100 μL of the treated culture is diluted 10-fold and 10.sup.5× and 100 μL is plated on 5-FU-supplemented LB agar plates and standard LB agar plates to quantify 5-FU resistant CFU and total CFU, respectively. Colonies are counted after a 24-hour incubation at 37° C. for the 5-FU plates and at 30° C. for the standard plates. After quantification, the plates are flooded with LB and scraped. These cell suspensions are transferred to culture tubes and incubated overnight at 30° C. Following this, genomic DNA is extracted from these mixed cultures and sent to Köln for deep amplicon sequencing.

Example 8: Testing in Bacillus subtilis with Different Genetic Target (pyrF or rpoB)

[0166] To verify that this technique works beyond the selected upp gene target, the process is adapted for pyrF gene (5-FOA resisresistance) or rpoB gene (Rifampin resistance).

Example 9: Targeted Mutagenesis for Functional Selection

[0167] This example describes combining catalytically inactive programmable DNA cleavage enzymes with DNA base modifying chemical mutagens to enrich mutagenesis in targeted regions of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS).

[0168] EPSPS is the enzyme that catalyzes the conversion of phosphoenolpyruvate and 3-phosphoshikimate to phosphate and EPSPS. This enzyme is inhibited by the competitive inhibitor glyphosate, which is used widely in agriculture as an herbicide. The structure of EPSPS has been determined and single point mutants within the active site that overcome glyphosate inhibition have been identified. Bacterial screens with E. coli have been developed that allow for selection of improved EPSPS variants in the presence of glyphosate (see Jin et al., Curr. Microbiol. 55:350 (2007)). Variant EPSPS enzymes have been generated by multiple methods, including untargeted methods such as error prone PCR or targeted approaches that are expensive and require highly-skilled researcher inputs to develop designs and molecular biology skills for saturation mutagenesis libraries.

[0169] DNA base modifying chemical mutagens in combination with catalytically inactive CRISPR associated protein/guide RNA complexes coupled together with activity selection, such as in a bacterial EPSPS functional selection assay, to enrich mutagenesis to a selected region of the enzyme EPSPS.

[0170] Cpf1 or Cas9 gRNAs targeting a specific region of the EPSPS enzyme, such as the residues lining the active site, are designed. This may require a synthetic gene containing proper PAM sites at the desired location(s).

[0171] E. coli expressing the EPSPS gene is transformed with dLbCpf1 or dSpCas9 and cognate gRNAs. The transformed cells are subsequently treated with a EMS, and mutagenized cells are placed under selection by glyphosate. Mutations accumulate at higher rates in the targeted region of EPSPS, and when placed under selection by glyphosate the recovery of resistance-conferring mutations derived from the targeted residues is increased.

Example 10: In Planta Targeted Gene Modification

[0172] Random chemical mutagenesis approaches to enhancing genetic diversity in plants requires balancing multiple factors for finding mutations in a candidate gene that include, mutation rate, viability and sterility after treatment, population size, the window of sequence evaluation, and others.

[0173] As the mutation rate decreases, the number of individuals required to screen to find a desired mutation increases exponentially. The local mutation rate induced by DNA base modifying chemical mutagens can be increased by utilizing sequence targeting enzymes (e.g., catalytically inactive RNA-guided endonuclease enzymes such as dLbCpf1 and dSpCas9). Once local mutation rates are increased, the number of individuals that need to be screened to find a desired mutation is reduced.

[0174] To enable this approach, the catalytically inactivated RNA-guided endonucleases and guide RNAs need to be present in the nucleus of a plant cell that will be treated with chemical mutagens. The catalytically inactivated RNA-guided endonuclease, gRNA, and EMS are titrated following standard procedures in the art to establish an initial kill-curve analysis for the dose and exposure times leading to a defined mortality (typically, 50% mortality is used in the art).

[0175] Targeted modification can be accomplished in multiple ways, including by expressing a catalytically inactivated RNA-guided endonuclease (e.g., dLbCpf1, dSpCas9) within the plant cell, either by co-delivering DNA or mRNA encoding the catalytically inactivated RNA-guided endonuclease or via stable transformation of the plant cells with the catalytically inactive RNA-guided endonuclease enzymes and/or gRNA. Following expression of the catalytically inactivated RNA-guided endonuclease and gRNA, EMS is applied using standard methods to induce targeted modifications of the target site.

[0176] An alternative approach for delivering a catalytically inactivated RNA-guided endonuclease and gRNA complex is to deliver the complex transiently as a ribonucleoprotein, which can be performed on a range of tissue types including leaves, pollen, protoplast, embryos, callus, and others. Following or concurrently with delivery, EMS is applied using standard methods to induce targeted modifications of the target site.

[0177] A number of seeds or regenerated plants are grown and screened for mutations in the targeted window using standard methods known in the art.

Example 12: Increasing Accessibility of DNA-Damaging Chemistries for Therapeutic Treatments

[0178] Direct chemical modification of DNA to interfere with normal DNA replication has been shown to be effective in cancer therapy. Cancer cells have relaxed DNA damage sensing/repair capabilities, which helps them achieve high replication rates and also makes them more susceptible to DNA damage.

[0179] The replication of damaged DNA increases the probability of cell death and has been the focus for anticancer compound development. The DNA alkylating-like platinum compound Cis-diamminedichloroplatinum(II) (cisplatin) forms DNA adducts with guanine and, to a lesser extent, adenine residues. When two platinum adducts form on adjacent bases on the same DNA strand they form instrastrand crosslinks. These intrastrand crosslinks block DNA replication and cause cell death if not repaired (see Cheung-Ong et al., Chem. Biol., 20:648-59 (2013)). These therapies are not without side effects and discovery efforts for cisplatin analogs are directed to reducing toxicity in nontargeted tissues (see Bruijnincx and Sadler, Curr. Opin. Chem. Biol., 12:197-206 (2008)).

[0180] The compositions and methods described herein may be used to increase the effectiveness of a non-targeted chemical DNA modifying therapeutic treatment. Not wishing to be bound by a particular theory, the DNA bases of essential genes can be made more available for chemical modification by the unwinding action of catalytically inactivated RNAguided endonuclease/guide complexes that unwind the DNA. The delivery of catalytically inactivated RNA-guided endonuclease/guide complexes to target cancer cells is an active area of development and routes for selective targeting of tumor cells could include, but not limited to oncolytic viruses or microinjection. These routes could be used for selective delivery of catalytically inactivated RNA-guided endonucleases (see Liu et al., J Control Release, 266:17-26 (2017)). The combined effect of selectively unwinding and making available the DNA targeted residues (by exposing the targeted base from within the more protected dsDNA helix) for chemical modification in cancerous cells may lower the total dosage of DNA damaging chemotherapeutic required to induce cell death in cancer cells. By lowering the total dosage of chemical therapeutic required, the adverse toxicity to non-target tissues would be expected to be reduced.

LEGEND TO FIGURES

[0181] FIG. 1: A plot of the rate of 5-FU resistant CFU relative to total CFU for each tested plasmid combination in Example 3, in both light cycled illumination (white bars) and dark conditions (black bars). Error bars represent standard deviation of triplicate samples.

[0182] FIG. 2: The relative resistance rates of tested fusion cargos in Example 5: light cycled illumination (white bars) and dark conditions (black bars). Error bars represent standard deviation of triplicate samples.

[0183] FIG. 3: The relative resistance rate of tested fusion cargos in Example 5, including an off-target control with Pp2FbFP_L30M targeted to amyE; light cycled illumination (white bars) and dark conditions (black bars). Error bars represent standard deviation of triplicate samples.

[0184] FIG. 4: The relative resistance rates of the tested guide combinations in Example 6; light cycled illumination (white bars) and dark conditions (black bars). Error bars represent standard deviation of triplicate samples.

TABLE-US-00007 List of Sequences SEQ ID NO: Identifier Remarks 1 dLbaCpf1_mOrange2_NA dLbaCpf1 fused to mOrange2 nucleic acid sequence 2 dLbaCpf1_mOrange2_AA dLbaCpf1 fused to mOrange2 amino acid sequence 3 pBV003dLbaCpf1_mOrange2_NA pBV003 plasmid encoding dLbaCpf1 fused to mOrange2 fluorescent protein nucleic acid sequence 4 pBV033dLbaCpf1_Pp2FbFP_L30M_NA pBV033 plasmid encoding dLbaCpf1 fused to Pp2FbFP_L30M nucleic acid sequence 5 pBV053_3Xupp_guides_NA pBV053 plasmid encoding guide array with 3 upp guides nucleic acid sequence 6 pBV041_amyE2_guide_NA pBV041 plasmid encoding amyE guide nucleic acid sequence 7 pcruppfor_NA Forward primer used to amplify upp region from B. subtilis 168 8 pcrupprev_NA Reverse primer used to amplify upp region from B. subtilis 168 9 sequppfor_NA Forward primer used to sequence upp region from B. subtilis 168 10 sequpprev_NA Reverse primer used to sequence upp region from B. subtilis 168 11 pBV030dLbaCpf1_SuperNova_NA pBV030 plasmid encoding dLbaCpf1 fused to SuperNova nucleic acid sequence 12 pBV031dLbaCpf1_tagRFP_NA pBV031 plasmid encoding dLbaCpf1 fused to tagRFP nucleic acid sequence 13 pBV032dLbaCpf1_SOPP3_NA pBV032 plasmid encoding dLbaCpf1 fused to SOPP3 nucleic acid sequence 14 pBV034dLbaCpf1_Chimera_NA pBV034 plasmid encoding dLbaCpf1 fused to chimera nucleic acid sequence 15 pBV040_amyE1_guide_NA pBV040 plasmid encoding amyE guide nucleic acid sequence 16 pBV049_upp1_guide_NA pBV049 plasmid encoding upp guide nucleic acid sequence 17 pBV050_upp2_guide_NA pBV050 plasmid encoding upp guide nucleic acid sequence 18 pBV051_upp3_guide_NA pBV051 plasmid encoding upp guide nucleic acid sequence 19 pBV055_5Xupp_guides_NA pBV055 plasmid encoding guide array with 5 upp guides nucleic acid sequence 20 pBV054_5XamyE_guides_NA pBV054 plasmid encoding guide array with 5 amyE guides nucleic acid sequence 21 dLbaCpf1 fused to Pp2FbFPL_30M amino dLbaCpf1 fused to Pp2FbFPL_30M amino acid acid sequence sequence, open reading frame of plasmid under SEQ ID NO: 4