Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing

Abstract

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

Fig. 1. Semi-rational engineering of Cas12i2 to improve indel activity in mammalian cells.

a Indel activity of wild-type Cas12i2 (WT) and SpCas9 expressed from a EFS promoter in HEK293T cells at 18 genomic target sites. Each circle represents a single target site, averaged from n = 2 replicates. Each bar represents the mean across 18 targets (ABR-001 in blue, SpCas9 and negative in gray). b Indel activity (relative to WT) of 14 single-substitution variants measured in HEK293T cells at two genomic target sites. Each circle represents indels for a single variant (n = 1). Black circles indicate the three mutations present in ABR-001. c Indel activity (relative to WT) of top three single substitutions and their combination variant, ABR-001. Bars represent mean of 2 targets and each circle represents n = 2 replicates at a single target site. d Indel activity of ABR-001 and SpCas9 expressed from a CMV promoter in HEK293T cells at 18 genomic target sites. Each circle represents a single target site, averaged from n = 2 replicates. Each bar represents the mean across 18 targets (ABR-001 in blue, SpCas9 and negative in gray). e. Indel size distribution comparison between SpCas9 and ABR-001 after genome editing of 18 target sites in HEK293T cells. The fraction of indels is analyzed by aggregating all indel reads, binning the reads by indel size (a minus value is deletion, a positive value is insertion). Circles represent average fraction of indels, binned by indel size, of 18 targets with n = 2 replicates (ABR-001 in blue, SpCas9 in gray). Source Data are provided as a Source Data file.

Fig. 2. ABR-001 is a specific nuclease for genome editing.

a Representative off-target discovery of ABR-001 and SpCas9 by TTISS at VEGFA Target 1. b–d TTISS analysis across 6 test loci for each of 3 genes. Teal (ABR-001) and blue (SpCas9) bars show average on-target indel activity for n = 2 replicates, each indicated by an outlined circle. Doughnut plots depict proportion of detected cleavage sites for each nuclease. Off-targets are shown as light gray wedges, while the on-target site is highlighted in teal (ABR-001) or blue (SpCas9). Centered numerical values represent fraction of on-target TTISS reads. Source Data are provided as a Source Data file.

Fig. 3. ABR-001 enables ex vivo editing of human primary cells.

a, b Indel activity (a) and viability (b) of human CD3 + T cells following delivery of ABR-001 RNPs with 4 different B2M-targeting guides (B2M 1–4) at decreasing concentrations (16 µM, 10 µM, 5 µM, and 2 µM). Cell viability was measured using flow cytometry at 7 days post-electroporation. Bars represent the average of 3 donors (circles), with error bars representing the s.d.. c Representative histograms show B2M protein level knockdown at 7 days post-electroporation with ABR-001 RNP with B2M 1 guide at decreasing concentrations (16 µM, 10 µM, 5 µM, and 2 µM). Y-axes represent count normalized to mode. d Indel activity of CD3+ T cells following delivery of 16 µM ABR-001 RNPs targeting TRAC and CIITA. Circles represent each of n = 2 bioreplicates. e Viability of CD3+ T cells following delivery of 16 µM ABR-001 RNPs targeting TRAC and CIITA. Circles represent each of n = 2 bioreplicates. f Representative histograms show protein level knockdown at 7 days post-electroporation for TRAC and CIITA (HLA-DR/DP/DQ readout). Y-axes represent count normalized to mode. g Indel size was compared between two independent experiments (BR1 and BR2) of ABR-001 RNPs targeting TRAC and CIITA. The color scale indicates the fraction of indel reads at a given indel size. h Indel rates (teal) and % GATAA disruption (black) of multiplexed ABR-001 RNPs (16 µM total concentration) or single SpCas9 RNP (5 µM concentration) targeting the BCL11A enhancer, 72 h post-delivery into human CD34+ cells. Bars represent mean and error bars represent s.d. of n = 3 replicates. i Indel rates (teal) and % GATAA disruption (black) of ABR-001 RNPs (20 µM total concentration) and SpCas9 RNP (5 µM concentration) after delivery and 20 days of maturation in erythroid differentiation media. Bars represent mean and error bars represent s.d. of n = 3 replicates. j Indel profiles of ABR-001 RNP (target 2 and multiplex target1+2) and SpCas9 RNP targeting the BCL11A enhancer in human CD34+ cells. The color scale indicates the fraction of indel reads at a given indel size. k Representative flow cytometric analyses of edited CD34+ cells after delivery and 20 days of maturation in erythroid differentiation media. Source Data are provided as a Source Data file.

Fig. 4. ABR-001 mediates highly efficient genome editing when delivered via AAV vector.

a Schematic of the AAV vector backbone for delivery of ABR-001 effector and a guide crRNA. b Indel rates at 72 h following transduction of ABR-001 and SaCas9 AAVs at increasing MOI in HEK293T cells. Teal (ABR-001) and gray (SaCas9) shaded bars represent the average of n = 2 bioreplicates, each indicated by an outlined circle. c Indel rates at two target sites measured daily for 7 days after transduction of HEK293T cells with ABR-001 AAVs at GC:Cell ratios of 1.6e4, 3.2e4, and 1.3e5. Shaded bars represent average indel rate of n = 2 bioreplicates for GC:Cell ratio of 3.2e4 and n = 1 bioreplicate for GC:Cell ratio of 1.6e4 and 1.3e5. Measurements for individual bioreplicates are indicated by an outlined circle. d ABR-001-AAV indel profiles across two targets and 2 MOIs over a time course of 7 days. The color scale indicates the fraction of indel reads at a given indel size. Source Data are provided as a Source Data file.