Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility

Abstract

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N₄AH and N₅HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N₃VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

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Fig. 1. Phage-assisted continuous and non-continuous evolution (PACE) to broaden the PAM compatibility of CjCas9.

a, Schematics of the PACE experiment. Selection phages (SP) infect E. coli host cells and use the hosts machinery to replicate and translate required phage genes. Instead of gene III, SP carries the ω-subunit-dCjCas9 fusion gene. Cas9 recognition of a PAM and protospacer sequence on the accessory plasmid (AP) allows recruitment of endogenous E. coli RNA polymerase via the ω-subunit and subsequent translation of gene III (pIII). Efficient recognition of the PAM and protospacer sequence results in infectious phages that are able to sustain in the lagoon, whereas weak or lack of recognition results in phage wash-out. The mutation rate during phage replication is controlled and elevated via arabinose-inducible expression of mutagenesis genes from a mutagenesis plasmid (DP6). b, Illustrative overview of 19 rounds of PANCE using accessory plasmids containing different PAM sites (according to IUPAC notation). Phages were increasingly diluted from one round to the next. Red indicates presence of phages, white indicates absence. c, Long-read sequencing of individual phages (n = 811) at 8 different time points during PACE. For each time point, the mutation frequency and amino acid position is shown. Amino acid positions substituted in more than 50% of phage genotypes are indicated.

Figure 2. Characterization of the PAM compatibility of evoCjCas9.

a, HT-PAMDA characterization of CjCas9 and evoCjCas9 illustrating their PAM preference at PAM positions 4 to 8. The log₁₀ rate constant represents the mean of two replicates against two distinct spacer sequences. b, Protein structure of CjCas9 with evoCjCas9 mutations introduced and highlighted (left, based on PDB: 5X2H). Detailed view illustrating differences between CjCas9 (top panels) and evoCjCas9 (bottom panels) residues interacting with PAM nucleotides of the target strand.

Figure 3. Indel formation rates of evoCjCas9 compared to CjCas9 and other commonly used RNA-guided endonucleases.

a, Illustration and size comparison of the expression vectors encoding for different Cas enzymes. b, Observed indel frequencies for CjCas9 and evoCjCas9 on 8 different PAM sites. c, Observed indel frequencies for different commonly used RNA-guided endonucleases and PAM relaxed Cas variants on different target sites with the most optimal PAM for each nuclease. Colors represent different timepoints. d, PAM frequency of the different RNA-guided endonucleases, illustrated as the probability of encountering a PAM on a randomly selected target site in a random DNA sequence (in %). Calculations are based on TTGAT for TnpB; TTTR for CasMINIv3.1; PAM sequences with a kinetic rate constant >10^-3 for CjCas9 variants; N₂GRRT for SaCas9; N₃RRT for SaKKH; N₂GG for SauriCas9; N₂RG for SauriKKH; N₄CC for Nme2Cas9; N₄C for eNme2-C.NR; NGG for SpCas9; NG for SpG and NR for SpRY. e, Mean indel frequencies of CjCas9-variants at on-target sites (0 MM) and potential off-target sites containing up to 5 mismatches (MM). f, Indel frequencies of evoCjCas9 at sites with dinucleotide mismatches normalized to the on-target site. Dinucleotides highlighted in red represent mismatch positions. PAM at position 23-30. Number above bars indicate amount of different target sites. g, Relative indel frequencies evaluated by HTS at off-target sites detected by CHANGE-seq for two sgRNAs. Boxplots in (b, c, e) represent the 25^th, 50^th, and 75^th percentiles. Whiskers indicate 5 and 95 percentiles. Numbers (n) above plots indicate number of biologically independent datapoints. Bars in (f, g) represent mean, with error bars representing standard error of the mean of indicated number of target sites (f) or biologically independent replicates (g, n=3, and n= 2 for OT2, 3, 4, 5 of CjCas9). b, c, e, f, g, Indels refer to insertion, deletions, or substitutions.

Figure 4. Base- and Prime editing with CjCas9 and evoCjCas9.

a, Illustration of expression vectors encoding for different CjCas9 BEs. b, Observed nucleotide transitions within the target sites with canonical and non-canonical PAMs. Bars represent mean of three independent biological replicates on N₄ACAC, N₄ATAC, N₄GTAC, and N₄GCAC (canonical) or N₄AAAC, N₄CAAC, N₄GAAC and N₄TAAC (non-canonical) PAM sites. Number of target sites (n) is indicated within each plot and bars represent mean of three independent biological replicates for each target site. Protospacer positions from -9 to 22 are shown. c, Illustration of expression vectors encoding for CjCas9 prime editors with the full length (top) or RnaseH depleted M-MLV reverse transcriptase (bottom). d, Comparison of CjCas9 or evoCjCas9 PEs installing an A to G transition at the AAVS1 locus. e, Mean editing efficiencies of CjCas9 or evoCjCas9-PE^ΔRnH on a selection of loci with canonical or non-canonical PAM sites. The loci of the target sites and type of edits are indicated. Bars (d, e) represent mean of n=2 (d) or n=3 (e) independent biological replicates in HEK293T cells, with error bars indicating standard error of the mean. f, Mean prime editing efficiencies and indel rates of biologically independent replicates (n=3) on 64 (canonical) and 172 (non-canonical) integrated target sites. Boxplots represent the 25^th, 50^th, and 75^th percentiles. Whiskers indicate 5 and 95 percentiles. CjCas9 in blue, evoCjCas9 in red (b, d − f).

Figure 5. In vivo genome editing with compact evoCjCas9 adenine and cytosine BE.

a, Schematic representation of single AAV vectors used for evoCjCas9 base editing. Elements are not illustrated to scale. b, Illustration of the experimental workflow for in vivo adenine or cytosine base editing at the Pcsk9 locus in the liver and of the Gpr6 locus in the brain. c, d, A-to-G editing at the targeted Pcsk9 splice site with different AAV concentrations, analyzed in isolated hepatocytes, whole liver samples, the tail (c, same legend as in h) and other tissues (d). e, Plasma PCSK9 levels relative to untreated control as determined by ELISA. ***P = 0.0006, **P = 0.0029, ***P = 0.0001, ****P < 0.0001 (left to right). f, Plasma LDL cholesterol levels. P = 0.2461, *P = 0.0278, *P = 0.027 (left to right). g, A-to-G editing at the targeted site in the Gpr6 locus (non-canonical CCGCCAAC PAM) in isolated brain tissues for CjCas9 and evoCjCas9. h, C-to-T editing at the targeted Pcsk9 site with evoCjCas9 cytosine base editor (eAID) in isolated hepatocytes, whole liver samples and the tail. i, Plasma PCSK9 levels relative to untreated control as determined by ELISA. *P = 0.0286. j, Plasma LDL cholesterol levels. **P = 0.0095. Means were compared using one-way ANOVA with Dunnett correction (e,f) or one-tailed t-test (Mann-Whitney) (i, j). Bars (c-j) represent mean with error bars representing standard error of the mean. Individual data points as black dots. Numbers (n) above plots indicate number of biologically independent datapoints. vg, vector genomes, chI, chimeric Intron, ICV, intracerebroventricular.