A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9

Home , Cas9

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9

2 sgRNA Libraries

3 ABSTRACT

4 CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have

5 enabled innovative CRISPR-based methods, such as the visualization of chromatin dynamics in

6 living cells. These libraries have the potential to be applied to a vast number of biological

7 systems and aid in the development of new technologies, but their synthesis is hindered by the

8 cost, time requirements, and technical difficulty of current sgRNA library generation methods.

9 Here, we describe SLALOM—a rapid enzymatic method for generating robust, variant-matched

10 sgRNA libraries from any source of DNA in under 3 hours. This method utilizes a custom

11 sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports

12 using a strand displacing polymerase. Using this method, we have constructed libraries targeting

13 the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its

14 potential to expand the reach of CRISPR technology and facilitate methods requiring custom

15 sgRNA libraries.

17 INTRODUCTION

18 Although originally harnessed for editing single genomic loci1,2, the Streptococcus

19 pyogenes CRISPR-Cas9 system has since been expanded to a number of high-throughput

20 molecular techniques utilizing oligonucleotide libraries encoding tens of thousands of unique

21 single-guide RNAs (sgRNAs) to target a large number of genetic loci3,4. These genome-wide

22 libraries have typically been cloned into lentiviral vectors for forward-genetic screening cells in

23 culture3,5–8. However, more recent studies have also adapted them for novel techniques, such as

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

24 chromosome painting9,10 and gene regulatory analysis11, expanding the potential for sgRNA

25 library-based methods.

26 A major hurdle in the application of the CRISPR sgRNA libraries is in generating the

27 library itself. Designing and chemically synthesizing a custom sgRNA library using microarray-

28 based or similar technologies12 can be expensive and time consuming and requires detailed

29 sequence information and specialized bioinformatics tools. Because they are based on a

30 reference sequence, chemically synthesized libraries can also differ significantly from the actual

31 subject of the study, limiting their application to a few model organisms with well-assembled

32 genomes and low rates of DNA sequence polymorphism. To address these issues, several

33 enzymatic approaches have been developed to create libraries from various DNA inputs10,13–15.

34 Using an enzymatic approach eliminates the need for subject-specific genomic sequence data,

35 greatly diminishes the number of sequence mismatches between the library and subject, and can

36 be synthesized at a fraction of the cost and time of chemically synthesized libraries. However,

37 these methods can be difficult to carry out, require large amounts of DNA, or result in libraries

38 where most of the sgRNAs are non-functional.

39 Here, we describe a streamlined enzymatic sgRNA library generation method that

40 produces high quality libraries from small amounts of input material. By designing a custom

41 sgRNA sequence containing a restriction endonuclease recognition sequence within the

42 repeat:anti-repeat duplex, we were able to develop a method that requires just two consecutive

43 sets of restriction digests and ligations. Additionally, our method constructs the library on the

44 surface of magnetic beads—minimizing the loss of material and simplifying purification between

45 steps. This method, which we have named SLALOM (sgRNA Library Assembly by Ligation On

46 Magnetic beads), can be carried out in a few hours and with less than 1 μg of DNA or cDNA as

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

47 input. Using SLALOM, we have generated sgRNA libraries from E. coli genomic DNA and

48 normalized cDNA from developing zebrafish hearts and show that libraries generated by this

49 method are effective in vivo through a fluorescent knockout screen in cell culture. Our data show

50 that SLALOM is a simple, rapid, cost-effective method for generating sgRNA libraries to be

51 used in a wide range of biological systems and techniques.

53 RESULTS

54 Method Design

55 To overcome the drawbacks of current enzymatic sgRNA library generation methods, we

56 developed the SLALOM method (outlined in Figure 1, complete protocol in Supplemental Note

57 1). First, the restriction enzyme HpaII (CCGG), which recognizes a DNA sequence containing a

58 PAM, is used to fragment the DNA (Step 1). HpaII was selected because its short palindromic

59 recognition sequence ensures high coverage and cuts adjacent to PAMs on both the forward and

60 reverse strands10. Next, an adapter is ligated to the ends of the digested fragments (Step 2). This

61 adapter contains a custom sgRNA scaffold sequence modified to incorporate an MmeI

62 recognition sequence and a long 5’ overhang. After ligation, the products are immobilized on

63 magnetic beads by hybridization of the long single-stranded overhang to a complementary

64 sequence affixed to the beads. After washing, the ligated DNA is digested by MmeI, which

65 cleaves at a fixed distance from its recognition sequence16, leaving 18/19 bp of the ligated DNA

66 sequence to form the sgRNA spacer (Step 3). After another wash, the fragments are ligated with

67 a second adapter containing a promoter region (e.g. T7 for in vitro transcription or U6 for in vivo

68 transcription) (Step 4). Finally, the beads are washed, and the completed library detached and

69 repaired using a strand-displacing polymerase (Step 5).

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

70 Modifications to the sgRNA Sequence

71 The method described here relies on a dual-role adapter that can function as a binding site

72 for MmeI and as a template for transcription of the sgRNA scaffold, thus requiring modifications

73 to the Cas9 binding region of the sgRNA without disrupting endonuclease activity. A previously

74 reported crystal structure of Cas9 in complex with the sgRNA revealed that the REC1 domain of

75 Cas9 interacts with the repeat:anti-repeat duplex of the sgRNA17. Functional experiments in this

76 same study showed that some mutations to the sequence of the duplex are permitted, while others

77 can diminish or completely inhibit the Cas9 activity. It was therefore unclear if an MmeI

78 recognition sequence could be incorporated into the repeat sequence without impairing the

79 function of the Cas9 complex.

80 Based on this information, we incorporated the MmeI recognition sequence (TCCRAC)

81 into the repeat sequence of the sgRNA at position 21 (directly after the spacer region) by making

82 two mutations, U24G and U25G, in the repeat sequence. We also made corresponding mutations,

83 A46C and A47C, in the anti-repeat sequence to maintain the repeat:antirepeat stem loop structure

84 (Figure 2A). Digestion of a 1000 bp DNA fragment in vitro with Cas9 and the wild type or

85 modified sgRNA showed cleavage efficiencies similar to or better than the unmodified sgRNA

86 with both an 18 bp and 20 bp spacer, indicating that endonuclease activity of the protein was

87 retained (Figure 2B, lanes 4 and 5).

88 The enzymes used to fragment the DNA prior to ligation with this adapter also produce

89 short 3’ overhangs not compatible with the wildtype Cas9 sgRNA sequence. Therefore, we next

90 engineered additional mutations to allow the sgRNA adapter sequence to hybridize with the

91 fragmented ends, eliminating the need for removal of the overhangs and increasing ligation

92 efficiency. This was accomplished by shifting the MmeI recognition sequence to position 23 and

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

93 modifying the sgRNA sequence by making mutations G21C, T22G, T23G, and A26G in the

94 repeat sequence and T45C, A48C and A49C in the anti-repeat sequence. Moving the MmeI site

95 to this position resulted in guide sequences of 18 and 19 bp (Figure 2A) instead of the standard

96 20 bp. However, digestion of the 1000 bp DNA fragment in vitro using an sgRNA with these

97 modifications again showed cleavage at the predicted location with similar efficiency to that of

98 the unmodified sgRNA using either a 20 bp or 18 bp protospacer (Figure 2B, lanes 6 and 7).

99 To confirm that sgRNAs with these modifications function in vivo, we tested an sgRNA

100 with these modifications and a spacer sequence targeting the zebrafish pigment gene gol

101 (slc24a5)18. Injection of single-cell zebrafish embryos with the modified sgRNA showed levels

102 of pigment loss at 48 hpf comparable to an unmodified sgRNA targeting the same spacer (Figure

103 2C). Together, these results demonstrate that modified sgRNAs can be used to guide Cas9

104 editing in vitro and in vivo.

105 Detaching Oligonucleotides with a Strand Displacing Polymerase

106 We also sought to simplify purification and handling of the library during construction by

107 incorporating magnetic bead purification into the SLALOM workflow. Constructing the library

108 on the surface of magnetic beads allows for rapid purification between steps without disrupting

109 the function of restriction enzyme or T4 DNA ligase activity19. To immobilize the modified

110 sgRNA adapter on the beads, a biotinylated ssDNA oligo was designed to hybridize with the 3’

111 end of the modified sgRNA adapter and a corresponding 19 base overhang was designed into the

112 3’ end of the adapter. The ssDNA oligos were then attached to streptavidin-coated paramagnetic

113 beads during the SLALOM protocol. Elution was accomplished by incubating the beads with a

114 strand-displacing polymerase, which elongates the 3' end of an oligo while displacing the pre-

115 existing strand, detaching the final library product from the beads (See Figure 1). Two strand

5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

116 displacing polymerases were tested: Bst 3.0, which is most efficient at temperatures near the

117 melting temperature of the bead-attached oligo, and Pol I, which is active at room temperature

118 and contains 5’-3’ exonuclease activity, which completely digests the oligo being displaced.

119 Oligo hybridization and elution assays showed that both enzymes were highly efficient (Figure

120 2), thus providing a simple technique to irreversibly detach oligonucleotides from solid supports

121 while simultaneously repairing the nicks caused by using unphosphorylated substrates in the

122 earlier ligation steps.

123 GFP Library and Fluorescent Knockout Screen

124 To assess functionality of a SLALOM generated library in cell culture, we used

125 SLALOM to create a library targeting the GFP coding region in GFP-LC3 Hela cells20. The GFP

126 construct in these cells contains four HpaII sites (CCGG) (Figure 3A). We PCR amplified the

127 GFP coding region (716 bp) to use as the SLALOM DNA input. In this iteration, the sgRNA

128 adapter was also modified to use a longer stem-loop, shown to potentially increase sgRNA

129 efficiency9, and both adapters incorporated Esp3I restriction sites for cloning into the

130 LentiCRISPRv2 plasmid21. Library sequencing confirmed complete coverage of the 8 expected

131 sgRNAs (Supplemental Figure 1). The cloned library was then packaged into lentivirus and used

132 to transfect HeLa GFP-LC3 cells. Sorting of the transfected and untransfected cells by FACS

133 showed that approximately 15% of the transfected cells lost GFP expression (Figure 3B,

134 Supplemental Figure 2), comparable to previous studies looking at CRISPR efficiency22.

135 Sequencing of the incorporated spacers and GFP mutations in each pool showed that all eight

136 potential spacers could be found in the GFP-negative pool and mutations occurred at all four

137 sites (Figure 3C). However, it was not possible to identify the mutation rate of each individual

138 sgRNA, as many of the common mutations deleted both cut sites within a pair (Figure 3D).

6 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

139 After establishing that all of the sgRNAs were active, we next analyzed their relative

140 mutation efficiency. Relative enrichment of each sgRNA in the GFP minus pool was determined

141 by normalizing the fractional counts from the sgRNA spacer sequencing results of the GFP

142 minus pool to their fractional counts in the GFP positive pool (Figure 4A). It should be noted that

143 this analysis is not completely analogous to enrichment analyses in genome-wide screens, as

144 there is no background gene set for normalization, but does show the relative efficiency of each

145 sgRNA in this library. These data showed that one sgRNA, a 19 bp sequence targeting the

146 negative strand of site 2, was more active than the others. There were also two sequences that

147 appeared to be significantly less effective than the average, an 18 bp sgRNA targeting site 3 and

148 an 18 bp sgRNA targeting site 4.

149 We initially hypothesized that this variance may be due to the fact that SLALOM does

150 not select for sgRNAs based on predicted efficiency. However, enrichment in the GFP-negative

151 pool did not show strong correlation with predicted efficiency score23 (Figure 4B, r2=0.003),

152 position in the gene (Figure 4C, r2=0.077) or length of the match (Figure 4D, r2=0.213). Overall,

153 these results show that the library can effectively target multiple sites and generate a measurable

154 enrichment of mutations causing a specific phenotype of interest, despite not using

155 bioinformatics analysis during the design the library. Furthermore, digestion with HpaII is

156 expected to generate an average of 13 sgRNAs per gene, much higher than current synthesized

157 libraries, providing additional opportunities to include highly efficient guides.

158 E. coli and Looping Zebrafish Heart Libraries

159 To test the method on a more complex DNA substrate, we isolated genomic DNA from

160 E. coli MG1655 and created an sgRNA library using SLALOM (Figure 5A). The E. coli genome

161 contains 24,311 HpaII sites. Because SLALOM produces two sgRNAs, one on the plus strand

7 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

162 and one on the minus strand, for each site, we predicted a full-coverage library would contain

163 48,622 sgRNA templates. High-throughput sequencing of the spacers was then conducted to

164 characterize the resulting sgRNA library. Sequencing results showed that the most common

165 spacer lengths were 18 and 19 bases (Figure 5B) and were evenly represented in the library

166 (Figure 5C). Finally, sites in the library covered 91.6% of the predicted HpaII sites and that

167 98.3% of the reads represented a PAM-adjacent spacer (Figure 5D), indicating that SLALOM

168 can be used to create high quality sgRNA libraries from a high molecular weight DNA input.

169 A clear advantage of SLALOM over chemically synthesized libraries is the ability to

170 create tissue or cell-type specific libraries without prior information on the genes expressed in

171 those cells. Thus, we next tested the ability of SLALOM to create an sgRNA library targeting all

172 of—but only—the genes expressed in the developing zebrafish heart (Figure 6A). Zebrafish

173 hearts were isolated from 48 hours post-fertilization (hpf) embryos for mRNA isolation. Because

174 genes are expressed at various levels, we next created normalized zebrafish heart cDNA by

175 subtractive hybridization24. The normalized cDNA library was confirmed by qPCR (Figure 6B)

176 and used as input for SLALOM.

177 Similar to the E. coli genome library, sequencing validation of the zebrafish heart library

178 found spacers that were predominately 18 or 19 bp long (Figure 6C). As sgRNAs in this library

179 were expected to only target expressed genes, we compared the library to a set of genes detected

180 in an existing zebrafish heart RNA-seq dataset25. These data showed the library targets at least

181 11,217 of the genes expressed in the heart (Figure 6D). Representation in the library is not

182 correlated with gene expression levels in the heart, confirming that cDNA normalization was

183 effective (Figure 6E). Thus, SLALOM can be used to target an sgRNA library to a specific

184 tissue.

8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

185 DISCUSSION

186 Generation of sgRNA libraries by chemical synthesis can be expensive, slow, and require

187 significant bioinformatic analysis of a known genome to identify suitable spacers. Because of

188 these limitations, synthesized libraries have been largely limited to a few well-established model

189 organisms with low DNA sequence polymorphism rates (such as inbred mice and human cell

190 lines) and complete genomes. However, these libraries cover only a small fraction of the

191 biological systems where they could be used. Wider adoption of CRISPR library methods in

192 non-traditional organisms and the development of new CRISPR-based technologies require the

193 development of more attainable library generation methods.

194 One alternative to chemical synthesis is enzymatic processing of DNA inputs into

195 sgRNAs. Several such methods have been proposed. Lane et al.10 digested DNA with restriction

196 enzymes containing a PAM in their recognition sequences and after ligation of a temporary

197 adapter used the restriction enzyme MmeI to capture the spacer before removing this adapter and

198 attaching an adapter containing the sgRNA sequence. This method resulted in an E. coli genome

199 library where 44% of the sequenced reads represented functional guides and 51% of the

200 predicted spacers were included. In contrast, the E. coli library presented here achieved results of

201 98.3% and 91.6%, respectively, for these same analyses. Arakawa, et al.13 also used a restriction

202 enzyme that cleaves outside of its recognition sequence to capture the spacer but selected for

203 PAMs by creating a cDNA library using a semi-random hexamer containing the dinucleotide

204 CC. This resulted in a library where 77.6% of the spacers were adjacent to the PAM. In contrast,

205 Cheng et al.14 and Köferle, et al.15 did not select for PAMs but produced libraries from

206 completely random fragments of a DNA sample. As a result, Cheng et al. produced dense

207 libraries targeting practically all valid PAMs and Köferle, et al. produced libraries where 23-30%

9 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

208 of the spacers were adjacent to PAMs and the spacer lengths distributed at about 27 bp.

209 However, both libraries contained many spacers that did not target PAM sequences. Finally,

210 library generation and screening have been combined in prokaryotes by transfecting the input

211 DNA directly into bacteria with a hyper-stimulated CRISPR system26. Thus, there was a wide

212 range of issues in the previous approaches, including complexity, reliability, difficulty, time

213 requirements, input amounts, and library quality and density.

214 SLALOM has several advantages over these methods, as it requires fewer steps and

215 smaller amounts of starting material while yielding sgRNA libraries in which almost all of the

216 final output contains functional guide sequences. Similar to the Lane et al. method described

217 above, SLALOM takes advantage of a restriction enzyme to determine the PAM, as this method

218 makes it possible to use any source of DNA input (e.g. genomic DNA, cDNA, or PCR products)

219 and to tune the density of the library by choosing enzymes with different recognition sequence

220 lengths and/or combining multiple restriction enzymes. However, unlike the Lane et al. method,

221 SLALOM uses a two-base overhang incorporated into the adapter sequence, eliminating the need

222 for mung-bean nuclease blunting of the digested fragments. Several previously reported

223 methods10,13,14 also temporarily ligate an intermediate adapter containing a recognition sequence

224 for a restriction enzyme that cleaves outside of this sequence to capture approximately 20 bp of

225 the fragment before removing the adapter. In SLALOM, this restriction enzyme site is directly

226 incorporated within the sgRNA template, eliminating the need for an intermediate adapter. Using

227 a single adapter also allows the reaction steps to occur while attached to a magnetic bead,

228 making purification between steps fast and simple and reducing material loss.

229 While SLALOM can already make high quality libraries, it also serves as a platform to

230 expand and diversify gene-targeting methods. For example, other restriction enzymes could be

10 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

231 used to create different sets of sgRNAs or in combination to increase library density. There

232 might also be ways to improve the mutation frequency, such as using a Type IIS enzyme with a

233 longer reach to generate sgRNAs that are 20-21 instead of 18-19 bases long. Based on previous

234 studies, this would likely increase cutting efficiency but could also decrease specificity27,28.

235 Finally, we made libraries using genomic DNA and normalized cDNA, but other sources of the

236 DNA input, such as pull downs to obtain DNA fragments bound by various proteins, could be

237 used as starting material. For example, conducting an RNA-PolII pulldown could make the

238 representation of genes actively transcribed in the tissue of interest more consistent than

239 normalized cDNA while still limiting the library to active genes, and pulldowns of other DNA-

240 binding proteins could also be used to examine subsets of enhancers or other non-coding regions,

241 especially if combined with repressor- or activator-bound Cas9 variants5,29–31.

242 One potential application of SLALOM-generated sgRNA libraries is in forward-genetic

243 screening. CRISPR screening in cell culture is already well established and has been shown to be

244 more effective than chemical mutagenesis or siRNA-based methods8,32. However, current

245 sgRNA libraries are generally synthesized to target all of the genes in the genome, which reduces

246 the number of times each gene can be targeted and increases the number of cells without

247 mutations in active genes, limiting the rate of gene discovery. However, enzymatic sgRNA

248 generation allows the library to be tailored to the genes expressed in the tissue and at the time

249 point of interest. For example, the zebrafish heart library reported here contains only sgRNAs

250 targeting the exons of genes expressed during heart looping morphogenesis. Thus, this method

251 will restrict mutations to genes involved in heart development, greatly improving the rate of gene

252 discovery and potentially expanding the number of biological systems that can be screened using

253 CRISPR technology beyond cell culture. Similar applications of SLALOM to novel experimental

11 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

254 designs and organisms will greatly increase the reach of CRISPR/Cas9 technology in a wide

255 range of fields.

256

257 EXPERIMENTAL PROCEDURES

258 Oligonucleotides and Bead Preparation

259 In vitro transcription of sgRNAs was accomplished by annealing and extending pairs of

260 oligos (see Supplemental Table 1 for oligos ordered from Integrated DNA Technologies

261 (Coralville, USA) or Eurofins (Louisville, KY)) by incubating at 66°C for 20 minutes to produce

262 double-stranded DNA templates containing a T7 promoter sequence, spacer sequence, and

263 sgRNA scaffold sequence and column purified. Either the MEGAscriptTM T7 Transcription Kit

264 (Thermo Fisher Scientific) or the HiScribeTM T7 Quick High Yield RNA Synthesis Kit (New

265 England Biolabs, Ipswich, USA) was used to transcribe the templates. In either case, 210-300 ng

266 of the template was used and incubated at 37°C for 1-2 hours. DNase I was then added, and the

267 reaction incubated for an additional 15 minutes at 37°C. The sgRNA was then purified using

268 either the RNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, USA) or by phenol-

269 chloroform extraction and ethanol precipitation.

270 Capture beads were prepared by resuspending 50 μL of streptavidin magnetic beads

271 (New England Biolabs, Ipswich, USA) in 20 μL containing 100 pmol of the biotinylated oligo

272 and incubating at room temperature for 15 minutes. The beads are then washed twice by

273 resuspending the beads in 50-100 μL of 1x Cutsmart® buffer. The ability of the biotinylated oligo

274 to bind to the magnetic beads was similar in both the recommended binding buffer as well as

275 CutSmart® buffer.

276

12 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

277 In vitro Digestion with Cas9

278 In vitro digestion of DNA fragments was accomplished by adding 200 ng of DNA, 70

279 pmol Cas9 Nuclease, S. pyogenes (1,000 nM) (New England Biolabs, Ipswich, USA), and 100

280 ng of the sgRNA in NEBuffer 3.1 (New England Biolabs, Ipswich, USA) for 20 minutes at 37°C

281 and 10 minutes at 65°C, followed by adding 1 μL of proteinase K (800 units/ml) (NEB) and

282 incubating at room temperature for 10 minutes before being run on a 1.7% agarose gel with a

283 100 bp ladder for 35 minutes.

284 DNA adapters were prepared by resuspending complimentary oligos at a final

285 concentration of 10 μM of each in CutSmart® buffer (New England Biolabs, Ipswich, USA). The

286 reaction was then heated to 98°C for 2 minutes then set to ramp from 85°C to 65°C for 1 hour

287 and finally from 65°C to 8°C for 30 minutes.

288 SLALOM Library Construction Method

289 A complete protocol for the SLALOM method can be found in Supplementary Note 1.

290 Briefly, DNA containing about 10 pmol of recognition sites for HpaII (CCGG) was added to a

291 50 μL reaction with a final concentration of 1x CutSmart® buffer and 10 units of HpaII. The

292 reaction was incubated at 37°C for 20 minutes and heat inactivated at 80°C for 20 minutes. 2,000

293 units of T4 DNA ligase, 10 pmol sgRNA adapter, and 50% PEG 6000 w/v were added to the

294 reaction at room temperature to bring the reaction to a total volume of 75 μL and 7.5% PEG. The

295 reaction was then incubated at room temperature for 20 min. The reaction was mixed with

296 capture beads for 15 minutes and the beads washed twice by exchanging the buffer for 50 μL 1x

297 CutSmart® buffer. The beads were then resuspended in 50 μL of 2 units MmeI and 50 µM S-

298 adenosylmethionine (SAM) in 1x CutSmart® buffer and incubated at room temperature for 20

299 min while keeping the beads in solution by occasionally pipetting. Beads were washed as

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

300 described above and resuspended in 50 μL of 1x CutSmart® containing 2,000 units of T4 and 30

301 pmol of the T7 adapter in 1x CutSmart®. The reaction was incubated for 20 minutes at room

302 temperature. The beads were washed and resuspended in 50 μL of 1x CutSmart® containing 10

303 units of DNA Polymerase 1 and 200 µM dNTPs in 1x CutSmart® and incubated for 5-10 minutes

304 at RT. A DNA Clean and Concentrate column (Zymo Research, Irvine, USA) was used to purify

305 the final library.

306 Zebrafish Embryo Injections

307 Zebrafish (Danio rerio) embryos were collected and injected at the single-cell stage with

308 Cas9:sgRNA Ribonucleoprotein (RNP) by incubating Cas9 (Integrated DNA Technologies,

309 Coralville, USA) with sgRNA in a 300 mM KCl solution for 5 minutes before injecting

310 approximately 1 nl of solution into each zygote. Injected embryos were kept at 28.5°C and after

311 2 days were visually examined for the extent of pigmentation. Photos were taken using an

312 Olympus SZX16 microscope.

313 Fluorescent Knockout Screening

314 GFP-LC3 HeLa cells stably expressing GFP were cultured in D10 medium (Dulbecco’s

315 modified Eagle’s medium (DMEM), Fetal bovine serum (10%), L-glutamine, penicillin,

316 Streptomycin) at 37°C with 5% CO2 and passaged every 3 days to maintain growth conditions.

317 Genomic DNA from these cells was isolated by collecting cells by centrifugation at 1000 g for 5

318 min and resuspension in 1x RIPA buffer containing Pronase at 37 °C for 10-15 min followed by

319 phenol chloroform extraction and ethanol precipitation. Genomic DNA was used as a template to

320 amplify the GFP coding region using the GFP forward and reverse primers to generate a 716 bp

321 fragment with 4 HpaII (CCGG) sites. This fragment was used as a substrate for SLALOM and

322 cloned into the lentiCRISPRv2 plasmid (Addgene, cat. no. 52961). The library digested with

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

323 Esp3I and purified using a DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, USA). The

324 plasmid was digested with Esp3I and NheI and the resulting 12,895 bp fragment was gel

325 extracted. Ligation using T4 DNA ligase of the two fragments was followed by transformation

326 into NEB® Stable Competent E. coli cells (New England Biolabs, Ipswich, USA) where 100 μL

327 was plated and 900 μL was used for an overnight culture in LB with 100 mg/ml Ampicillin. The

328 overnight culture was used to inoculate a 500 mL LB with the antibiotic and the plasmid DNA

329 was isolated using NucleoBond Xtra Midi EF (Macherey-Nagel, Dueren, Germany). Sequence

330 flanking the spacer sequences were amplified and were sequenced. Following validation of the

331 library, it was packaged into lentivirus by VectorBuilder (Chicago, USA).

332 To calculate the multiplicity of infection (MOI), two 6 well dishes were seeded at

333 approximately 20% confluency and allowed to attach to the plates. Cells from the first dish were

334 transduced with increasing amounts of the virus in media containing 6 μg/mL Polybrene. After 2

335 days of growth, media was replaced with media containing 8 μg/mL Puromycin for two

336 additional days until all cells in the control well had died and the surviving cells were counted,

337 and the ratio was used to calculate a curve. Cells infected at an estimtaed MOI of 0.3 and sub-

338 cultured to obtain enough cells for sorting. After 10 days, cells were sorted based on

339 fluorescence using a FACS Aria Fusion and the sorted cells were cultured separately. Genomic

340 DNA from these two populations as well as from uninfected cells was isolated and the GFP

341 coding region was sequenced. The spacers from these two populations were also sequenced.

342 E. coli Genomic DNA Isolation

343 Genomic DNA from Escherichia coli MG1655 was isolated using the PowerLyzer®

344 UltraClean® Microbial DNA Isolation Kit from Qiagen (Hilden, Germany). Bacterial cells were

345 harvested and lysed using glass microbeads. Final product was run on a gel and a clear band of

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

346 high molecular weight was observed. The genomic DNA was used as a substrate for SLALOM

347 and the resulting library was sequenced.

348 Developing Zebrafish Heart mRNA Extraction

349 Zebrafish hearts were isolated as published previously33. Briefly, about 200

350 Tg(myl7:GFP) zebrafish embryos were collected at 48 hours post-fertilization and placed in

351 media containing tricaine. Embryos were resuspended in L-15 media with 10% FBS and the

352 tissue of the embryos was disrupted by passing them through a 19G needle. Intact hearts were

353 isolated under a fluorescent microscope with a pipette and after being washed were resuspended

354 and homogenized in TRI-Reagent® (Zymo Research, Irvine, USA). Zebrafish heart mRNA was

355 then purified using the Direct-zolTM RNA Kit (Zymogen Research, Irvine, USA).

356 cDNA Library Synthesis and Normalization

357 The SMART® cDNA Library Construction Kit (Clontech) was used to reverse transcribe

358 the zebrafish heart RNA. An alternative oligonucleotide that lacked an HpaII binding site was

359 used in place of the 5' oligo provided by the kit to prevent gRNA creation to the adapters in the

360 cDNA library (Supplementary Table 1). The cDNA library was then normalized using the

361 Trimmer-2 cDNA normalization kit (Evrogen, Moscow, Russia).

362 High-throughput Sequencing and Data Analysis

363 High throughput sequencing of the libraries was carried out at the University of Utah

364 Shared Genomics Resource on an Illumina HiSeq 2500. PhiX DNA was added to each library

365 before sequencing to allow for more diversity during the initial reading process. Custom scripts

366 were generated to extract the targeting region of each sgRNA template and analyze the data

367 (Supplemental Notes 2 and 3). Because the length of the library was 128 bp, but the read length

368 was only 50 bp, about half of the reads were discarded because they did not contain the spacer.

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

369 The extracted spacer sequences were then aligned to either the Escherichia coli MG1655 genome

370 or the zebrafish GRCz11 genome. Statistical analysis was then used to determine the coverage of

371 each library.

372 Confirmation of the GFP mutation rates was conducted by high-throughput sequencing

373 using the Genewiz Amplicon-EZ service (South Plainfield, USA) and analyzed using custom

374 scripts in R based on the Cris.py pipeline34 (Supplemental Notes 4 and 5). Briefly, reads

375 containing complete PCR products were selected and searched for a 30 bp sequence spanning

376 each HpaII site. Reads were categorized by whether they contained wild type sequences at each

377 site. Reads with identified mutations at each site were then tabulated to find the mutation

378 frequency at the site and the most common mutations. Reads with large deletions that spanned

379 multiple HpaII sites were categorized separately as “Multiple”.

380

381 DISCLOSURES

382 IACUC Approval

383 All animal studies were approved by the Brigham Young University Institutional Animal Care

384 and Use Committeee under protocol number 18-0704.

385 Data availability

386 All high-throughput sequencing data used in this study are available through the Sequence Read

387 Archive (Project ID: PRJNA642300).

388 Acknowledgments

389 The Hela cells used here were a generous gift from Joshua L. Anderson. We would also like to

390 thank Marc Hansen, Brent Nielsen, and Jeffery Barrow for their critical reading of the

391 manuscript.

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

392 Competing Interests

393 J.H. and J.Y. are inventors of US Patent No. 10,669,539 and are co-founders of Pioneer Biolabs,

394 LLC.

395 Funding

396 This work was supported in part by NIH 1R15HD098969 (J.T.H.) and UM1 HL098160 (HJY),

397 and sequencing was supported by a core facilities support grant to CCHCM (U01HL131003),

398 from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the

399 authors and does not necessarily represent the official views of the National Institutes of Health.

400

401 FIGURE LEGENDS

402 Figure 1. Schematic of the sgRNA Library Synthesis Method. Each step of the process is

403 represented by a black arrow. Restriction enzyme binding sites are shown in black. Magnetic

404 beads are represented as orange circles. (Step 1) DNA from an arbitrary source is fragmented by

405 a restriction enzyme such as HpaII (CCGG) that contains the canonical PAM (NGG) in its

406 recognition sequence. (Step 2) An adapter (blue) encoding a modified sgRNA sequence is ligated

407 to the DNA fragments. The adapter is unphosphorylated, contains an MmeI recognition

408 sequence, has a two base-pair, single-stranded overhang compatible with the fragmented DNA,

409 and a long single-stranded overhang capable of hybridizing to a single-stranded oligo. (Step 3) A

410 single-stranded capture oligonucleotide attached to a magnetic bead at the 3’ end is used to

411 immobilize the adapter to magnetic beads through hybridization and excess DNA is washed

412 away before digestion with MmeI to capture the spacer sequence. (Step 4) An unphosphorylated

413 adapter containing the T7 promoter sequence (green) and a 3’ overhang containing all possible

414 dinucleotides is ligated to the cleaved spacer sequence. (Step 5) The nicked library is detached

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

415 from the beads by a strand displacing polymerase (yellow) and nicks are simultaneously repaired

416 by the same mechanism. The detached double stranded oligonucleotides constitute an sgRNA

417 library containing spacers targeting the DNA source and can be transcribed in vitro or cloned

418 into plasmids for downstream applications.

419

420 Figure 2. Incorporation of the MmeI Recognition Sequence into the Repeat:Anti-Repeat

421 Duplex and Elution with a Strand Displacing Polymerase. A) Modifications were made to the

422 Repeat:Anti-Repeat Duplex of the sgRNA sequence to insert a single MmeI site that cleaves 18-

423 20 bp upstream of the sgRNA recognition sequence. Mutations are shown in red. The MmeI

424 binding sites are shown in dark gray boxes. The MmeI cut site is represented with a triangle. B)

425 in vitro Cas9 digestion of a 1000 bp DNA fragment with each sgRNA variant. C) Injection of

426 zebrafish embryos with Cas9 and the sgRNA variant targeting the gol gene compared to an

427 uninjected control (minimum n per pool = 150) D) Detachment of the sgRNA scaffold from

428 magnetic beads using Bst 3.0. E) Detachment of the sgRNA scaffold from magnetic beads using

429 Pol1.

430

431 Figure 3. GFP Knockout Screen. A) Genomic DNA was isolated from HeLa cells stably

432 expressing GFP-LC3. The four HpaII sites within the GFP ORF and the predicted sgRNAs are

433 labeled. B) FACS sorting of transfected and untransfected cells. GFP fluorescence is on the x-

434 axis and counts are on the y-axis. C) Mutation frequency at each of the HpaII sites based on

435 high-throughput sequencing of the GFP locus. D) The most common variants for each of the four

436 HpaII sites.

437

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

438 Figure 4. Relative Efficiency of sgRNAs in the GFP Library. A) Relative efficiency of each

439 sgRNA measured by fold enrichment in the GFP-minus HeLa cell pool compared to the GFP-

440 positive pool. B) Comparison of relative efficiency in the screen vs. predicted efficiency scores23

441 for the locus. C) Comparison of relative efficiency in the screen vs. location in the GFP coding

442 region. D) Comparison of relative efficiency vs. length of the match between the spacer and

443 protospacer.

444

445 Figure 5. High-throughput sequencing of a SLALOM-generated sgRNA library to the E.

446 coli MG1655 genome. A) Schematic diagram of the library generation process. B) Histogram

447 showing the distribution of spacer lengths within the library. C) Histogram showing

448 representation of each sgRNA target in the library. D) Analysis showing the coverage and purity

449 of the library.

450

451 Figure 6. High-throughput sequencing of a SLALOM-generated sgRNA library to

452 normalized cDNA extracted from a 48 hpf zebrafish heart. A) Schematic diagram of the

453 library generation process. B) Results of qPCR analysis on five genes with varying expression

454 levels in the endogenous tissue before and after cDNA normalization. C) Histogram showing the

455 distribution of spacer lengths within the library. D) Venn diagram of the genes targeted by at

456 least one spacer in the library compared to genes detected in the 48 hpf zebrafish heart by RNA-

457 seq. A threshold of 1 read per gene was used to designate both genes and spacers as “detected”.

458 E) Comparison of the total number of spacer reads vs. expression level in an RNA-seq dataset

459 for 48 hpf zebrafish hearts.

460

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

461 REFERENCES

462 1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive

463 bacterial immunity. Science (80-. ). 337, 816–821 (2012).

464 2. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science (80-. ).

465 339, 819–823 (2013).

466 3. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells.

467 Science 343, (2014).

468 4. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using

469 the CRISPR-Cas9 system. Science (80-. ). 343, 80–84 (2014).

470 5. Sanson, K. R. et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple

471 modalities. Nat. Commun. 9, 5416 (2018).

472 6. Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome

473 using CRISPR-Cas9. Nat. Biotechnol. 34, 192 (2016).

474 7. Viswanatha, R., Li, Z., Hu, Y. & Perrimon, N. Pooled genome-wide CRISPR screening

475 for basal and context-specific fitness gene essentiality in Drosophila cells. Elife 7, (2018).

476 8. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera, M. D. C. & Yusa, K. Genome-wide

477 recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA

478 library. Nat. Biotechnol. 32, 267–273 (2014).

479 9. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized

480 CRISPR/Cas system. Cell 155, 1479–1491 (2013).

481 10. Lane, A. B. et al. Enzymatically Generated CRISPR Libraries for Genome Labeling and

482 Screening. Dev. Cell 34, 373–378 (2015).

483 11. Klann, T. S. et al. CRISPR-Cas9 epigenome editing enables high-throughput screening for

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

484 functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568

485 (2017).

486 12. Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: Technologies and

487 applications. Nature Methods vol. 11 499–507 (2014).

488 13. Arakawa, H. A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing

489 of any organism. Sci. Adv. 2, e1600699 (2016).

490 14. Cheng, J. et al. A Molecular Chipper technology for CRISPR sgRNA library generation

491 and functional mapping of noncoding regions. Nat. Commun. 7, (2016).

492 15. Köferle, A. et al. CORALINA: a universal method for the generation of gRNA libraries

493 for CRISPR-based screening. BMC Genomics 17, 917 (2016).

494 16. Morgan, R. D., Dwinell, E. A., Bhatia, T. K., Lang, E. M. & Luyten, Y. A. The MmeI

495 family: type II restriction-modification enzymes that employ single-strand modification

496 for host protection. Nucleic Acids Res. 37, 5208–21 (2009).

497 17. Nishimasu, H. et al. Crystal Structure of Cas9 in Complex with Guide RNA and Target

498 DNA. Cell 156, 935–949 (2014).

499 18. Burger, A. et al. Maximizing mutagenesis with solubilized CRISPR-Cas9

500 ribonucleoprotein complexes. Development 143, 2025–37 (2016).

501 19. Pengpumkiat, S., Koesdjojo, M., Rowley, E. R., Mockler, T. C. & Remcho, V. T. Rapid

502 synthesis of a long double-stranded oligonucleotide from a single-stranded nucleotide

503 using magnetic beads and an oligo library. PLoS One 11, e0149774 (2016).

504 20. Mizushima, N., Yoshimori, T. & Levine, B. Methods in Mammalian Autophagy Research.

505 Cell vol. 140 313–326 (2010).

506 21. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

507 CRISPR screening. Nat. Methods 11, 783–784 (2014).

508 22. Jin, J. et al. An improved strategy for CRISPR/Cas9 gene knockout and subsequent

509 wildtype and mutant gene rescue. PLoS One 15, 1–21 (2020).

510 23. Kim, H. K. et al. SpCas9 activity prediction by DeepSpCas9, a deep learning–based

511 model with high generalization performance. Sci. Adv. 5, eaax9249 (2019).

512 24. Diatchenko, L. et al. Suppression subtractive hybridization: A method for generating

513 differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad.

514 Sci. U. S. A. 93, 6025–6030 (1996).

515 25. Hill, J. T. et al. Heart morphogenesis gene regulatory networks revealed by temporal

516 expression analysis. Development 144, 3487–3498 (2017).

517 26. Jiang, W., Oikonomou, P. & Tavazoie, S. Comprehensive Genome-wide Perturbations via

518 CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity. Cell 180, 1002-

519 1017.e31 (2020).

520 27. Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational

521 checkpoint between DNA binding and cleavage by CRISPR-Cas9. Sci. Adv. 3, eaao0027

522 (2017).

523 28. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas

524 nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

525 29. Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-

526 mediated gene repression and activation. Elife 5, (2016).

527 30. Gilbert, L. A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and

528 Activation. (2014) doi:10.1016/j.cell.2014.09.029.

529 31. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

530 Cas9 complex. Nature 517, 583–588 (2015).

531 32. Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and

532 metastasis. Cell 160, 1246–1260 (2015).

533 33. Burns, C. G. & MacRae, C. A. Purification of hearts from zebrafish embryos.

534 Biotechniques 40, 274–282 (2006).

535 34. Connelly, J. P. & Pruett-Miller, S. M. CRIS.py: A Versatile and High-throughput Analysis

536 Program for CRISPR-based Genome Editing. Sci. Rep. 9, 1–8 (2019).

537

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Figure 2 available under aCC-BY-NC-ND 4.0 International license. a b A Repeat B Modified sgRNAs GUUUUAGA--GCUA sgRNA Repeat G Modified sgRNAs A |||||| ||||BA AA No sgRNAWT (20 nt)WT (18 nt)MmeI (20MmeI nt) (18MmeI nt) + HpaIIMmeI (20 + HpaIInt) (18 nt) sgRNA GUUUUAGA--GCUAU C-GGAAG UAAAAUUGAACGAU |||||| |||| A | |||A U C-GGAAUAAAAUUGAACGAUA A A | ||| GUCCGUUAUCAACUUG GUCCGUUAUCAACUUG A A ||||| ||||| Anti-Repeat Anti-Repeat AGCCACGGUGAAA A A G ||||| AGCCACGGUGAAA UCGGUGCUUUU 3’ G ||||| 1 kb Ladder (-) sgRNA Standard (20 nt)MmeI (20 nt) Standard (18 nt)MmeI + HpaII (18 nt) UCGGUGCUUUU 3’ 1 kb Ladder (-) sgRNA Standard (20 nt)MmeI (20 nt) Standard (18 nt)MmeI + HpaII (18 nt) Standard (20 nt) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG...(+45 nt) 2.0 - MmeI (20 nt) ------GTTGGA------CC------... 1653 bp WT 1.5 - Standard (18 nt) ------... 1.0 - 939 bp MmeI + HpaII (18MmeI nt) ------CCGGTTGGA------C--C------... 714 bp Standard (20 nt) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAAT0.5 - AAGG...(+45 nt) MmeI + HpaII 2.0 - MmeI (20 nt) ------GTTGGA------CC------... 1.5 - 1653 bp Standard (18 nt) ------Repeat Anti-Repeat------...% cut: 0 46 75 62 72 65 59 Repeat Anti-Repeat 1.0 - 939 bp MmeI + HpaII (18 nt) ------CCGGTTGGA------C--C------... 714 bp c 0.5 - Uninjected Control Standard sgRNA slc24a5 (gol) MmeI + HpaII sgRNA slc24a (gol) d C L Enz. – L Enz. –

Repeat Anti-Repeat

C Uninjected Control Standard sgRNA slc24a5 (gol) MmeI + HpaII sgRNA slc24a (gol) Bst 3.0 Pol1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.175117; this version posted July 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 1 a Original Library GFP Positive Pool GFP Minus Pool

1% 1% 2% 10% 8% 4% 5% 6% 4% 5% 6% 7% 4% 2% 21% 25% 17% 10% 1% 4% 16% 3% 3% 1% 1% 4% 1% 16% 2% 21% 2% 1% 13% 3% 23% 12% 0% 6% 4% 8% 2% 3% 2% 4% <1% 1% 1% 1%

b Original Library GFP Plus Pool GFP Minus Pool

11% 8% 8% 6% 7% 23% 5% 5% 6% 8% 9% 5% 24% 29% 4% 10% 17% 19% 14% 10%

23% 9% 16% 24%

Supplemental Figure 1. Relative representation of sgRNAs. The overall library, GFP Positive Cells after FACS sorting, and GFP Minus Pool after FACS sorting were sequenced to identify the relative representation of each potential sgRNA. BD FACSDiva 8.0.1

BD FACSDiva 8.0.1 Supplemental Figure 2

a BD FACSDiva 8.0.1

Global Sheet2 Printed on: Mon Jul 20, 2020 03:33:36 MDT

BD FACSDiva 8.0.1 b

Global Sheet2 Printed on: Mon Jul 20, 2020 03:33:26 MDT

Supplemental Figure 2. Raw FACS Results. A) Raw FACS results for live cell gating for untransfected cells. B) Raw FACS results for live cell gating for transfected cells. FSC-A = Forward Scatter Area, FSC-H = Forward Scatter Height, SSC-A = Side Scatter Area.

Global Sheet2 Printed on: Mon Jul 20, 2020 03:33:36 MDT

Global Sheet2 Printed on: Mon Jul 20, 2020 03:33:26 MDT