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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 16 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 52 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).