Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5′ homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.

通过 CRISPR 编辑的小鼠 B 细胞过继移植来表达人抗体可变链，可以帮助评估候选疫苗并开发更好的抗体疗法。然而，当前的编辑策略破坏了重链位点，导致低效的体细胞超突变，而没有功能亲和力成熟。在这里，我们表明，通过在每个位点使用单个 Cas12a 介导的切割和与远端 V 片段互补的 5' 同源臂，直接同时用人抗体的重链和 kappa 链替换重组的小鼠重链和 kappa 链，可以保留这些关键的 B 细胞功能。以这种方式编辑的细胞表达人类免疫缺陷病毒 1 型 (HIV-1) 广泛中和抗体 10-1074 或 VRC26.25-y，在接种疫苗的小鼠中发生强烈超突变并产生有效的中和血浆。从小鼠中分离出的 10-1074 变体比野生型 10-1074 更有效地中和了一组 HIV-1 分离株，同时保持了其低多反应性和长半衰期。我们还使用该方法来提高抗 SARS-CoV-2 抗体针对最新 Omicron 菌株的效力。在其天然位点编辑的 B 细胞的体内亲和力成熟可能会促进广泛、有效和生物可利用的抗体的开发。